Atrial fibrillation (AF) is a complex cardiac arrhythmia in clinical practice with increasing incidence. However, the effects of statins on patients with AF are not quite clear.To investigate the protective effect of calcium channel blocker (CCB) and valsartan combined fluvastatin on hypertension (HTN) patients with nonpermanent AF.In three and a half years, 189 cases of patients diagnosed as HTN combining nonpermanent AF by eight medical centers, were recruited and randomly assigned to four groups with varied treatments: CCB group; CCB + statin group; valsartan group; and valsartan + statin group. The four groups were followed up for 24 months. The 7‐day Holter ultrasound echocardiography (UCG) and biochemical indexes were completed at preset time nodes respectively.P < .05 or P < .01). Furthermore, the improvement trend of CCB + statin group and valsartan + statin group was more obvious.After 24 months of follow‐up, 178 patients completed the study. Compared with CCB group, the blood lipid level, inflammatory index, ultrasonic index and electrocardiographic measurement results of CCB + statin group, valsartan group and valsartan + statin group were improved in different degrees and had statistical significance (The results indicated that valsartan can reduce AF load and recurrence rate, and delay the progression of nonpermanent AF to permanent AF in multiple ways, and the effect of combination of valsartan and fluvastatin is more significant. These results provide a new direction for the integrated upstream control strategy of AF. We obtained the written informed consent from each patient before the trial. Before performance of this trial, the study protocol, informed consent of patients, case report form and other essential materials have been submitted to the ethics committee of the Second Hospital of Tianjin Medical University (IEC). The study has been reviewed and approved by IEC  and has been registered in the Chinese Clinical Trial Registry (registration number: ChiCTR‐TRC‐12002642). The article for relevant trial scheme has been published.2.2We recruited 189 patients who have been diagnosed as HTN combined with nonpermanent AF by outpatient or inpatient department from eight medical centers in China between February 2013 to July 2016. The eight medical centers are listed as follows: The Second Hospital of Tianjin Medical University, People's Hospital of Nankai University, Tianjin Dongli Hospital, Tianjin Jinghai Hospital, Tianjin Beichen Hospital of Tradition Chinese Medicine, Tianjin Port Hospital, First Hospital of Qinhuangdao and Tangshan Gongren Hospital.Entry criteria were (1) HTN, defined as the systolic blood pressure (SBP) ≥ 140 mmHg (1 mmHg = 0.133kpa) and/or diastolic blood pressure (DBP) ≥ 90 mmHg continuously measured twice in a s resting state, or the patient who has blood pressure controlled as less than 180/110 mmHg by using antihypertensive drugs since his/her clear history of HTN; (2) at least two symptomatic AF attacks recorded by electrocardiogram (ECG) within 6 months before enrollment, and the duration of AF was less than 1 year. In addition, at the time of enrollment, abnormal AF rhythm could be converted to normal sinus rhythm independently or by medical intervention. However, drug and/or electrical cardioversion must be performed between 6 months and 8 weeks before enrollment, and no cardioversion was undertaken in the last 8 weeks; (3) did not take angiotensin converting enzyme inhibitors (ACEIs), angiotensin receptor blockers (ARBs) and/or statins for more than 2 weeks before enrollment, or taking the above drugs, but can withstand the washout period of more than 2 weeks after discontinuation; (4) aged between 25 to 79 years; and (5) signing informed consent voluntarily.Exclusion criteria were (1) permanent AF or persistent AF lasting for more than 1 year; (2) unstable angina pectoris (UAP) with poor controlled; (3) severe left main coronary artery disease confirmed by coronary angiography (CAG); (4) acute myocardial infarction (AMI) within 3 months; (5) grade III or IV HF (NYHA heart function grade); (6) valvular disease with indications of surgical operation or interventional therapy; (7) stroke or transient ischemic attack (TIA) within 3 months; (8) thyroid disease with uncontrollable complications ; (9) severe liver or renal function damage defined as alanine aminotransferase (ALT) more than 80 U/L, aspartate aminotransferase (AST) more than 80 U/L or creatinine (Cr) more than 1.5 mg/dL; (10) severe hyperlipidemia needed essential treatment with statins/bates defined as total cholesterol (TC) more than 6 mmol/L or low density lipoprotein cholesterol (LDL‐C) more than 3.36 mmol/L; (11) Other ARB, ACEI and/or statins have been taken and cannot be washed for more than 2 weeks; (12) contraindications for statins and/or ARBs; (13) pregnant/planned pregnant women or lactating women; (14) the possibility of poor compliance or noncooperation during holistic follow‐up; and (15) inappropriate age.2.3Participants were randomly assigned to be treated either with dihydropyridine CCB , CCB combined with fluvastatin , valsartan , or valsartan combined with fluvastatin . Patients' medical history, medication history  and results of physical examination were recorded before drug intervention. Furthermore, resting ECG, 7‐day electrocardiogram (7‐day Holter), UCG, related blood routine, whole blood biochemistry and other laboratory examinations were also tested. In this study, valsartan  was recommended to take in the morning every day as an initial dose of 80 mg; fluvastatin  had an initial dose of 40 mg, once a day, before going to bed; and dihydropyridine CCB was only allowed to use amlodipine, nifedipine or felodipine. After patients entering the group, the dosage of each group could be adjusted according to the clinical situation. However, all the patients must meet the standard of blood pressure control as 140/90 mmHg. The recommended dosage of valsartan and fluvastatin were ≥ 80 mg and ≥ 40 mg, respectively.2.4am AF attack time was selected to measure the f‐f interval. In detail, successive 20 f‐f intervals that from the second or the third f wave after either QRS wave were measured in order to avoid the influence of QRS complex wave, T wave and U wave. Furthermore, in the same period, lead III was selected to measure the amplitude of wave. Similarly, consecutive 20 f waves were recorded. After that the average of these measured value was taken for comparison. It is worth noting that the f‐f interval refers to the distance between the starting point of the previous f wave and the starting point of the next f wave while f wave amplitude means the distance from the upper edge of f‐wave peak to the upper edge of the valley. At last, AF load was defined as the total time of AF attack recorded in 7‐day Holter as a percentage of the total recording time.Subjects filled in their case report form at the beginning of the trial, after that their gender, age, body mass index , history of diabetes, stroke, smoking or drinking, UCG, resting ECG, 7‐day Holter as well as blood routine, blood lipid, myocardial enzyme, liver and renal function and other blood biochemical laboratory test results were recorded in detail. The combined medication and adverse events were observed within 24 months of follow‐up. During this period, all the selected patients were followed up once every 3 months to review blood pressure, ECG, and cardiac function classification (NYHA). In addition, patient logs, disease changes, new combined drugs, adverse events and doctor's recommended prescriptions were also recorded. At the 24th month, UCG, related blood routine test, whole blood biochemistry and other laboratory tests were reexamined for comparison. Holter was reexamined at the 6th, 12th and 24th months. For the 7‐day Holter, 22:00‐06:00 2.5t test and multiple comparisons of groups were made by one‐way ANOVA followed by Bonferroni correction. P values below .05 were considered significant.SPSS 20.0 was utilized to analyze the data. The measuring data of near normal distribution were described by mean standard ± deviation and the counting data was expressed by percentage. The comparison of changes in before and after treatments in each group was made by Student's 3Eleven participants quit the study during the observation and treatment. In CCB group, there were six patients withdrew from the trial because of intolerable edema after taking CCB drugs (n = 2), prolonged QT interval more than 0.5 s (n = 2) or severe sinus bradycardia (n = 2). Only one patient withdrew since liver dysfunction in CCB + statin group. In valsartan group, two cases quit due to poor blood pressure control that needed to use other antihypertensive drugs except ARB (n = 1) or abnormal renal function (n = 1). In valsartan + statin group, one patient quit because of AMI while the other patient withdrew since severe sinus bradycardia. After the above patients were excluded, 41, 45, 44, and 48 patients in CCB group, CCB + statin group, valsartan group and valsartan + statin group were available for analysis. According to the rules of Statistics, the proportion of exfoliated cases was acceptable so that it would not make a significant negative impact on the research results.3.1P > .05). No significant difference was found in mean SBP and DBP among the four groups (P > .05). In addition, at the end of follow‐up, the blood pressure of all the four groups was lower than that before treatment. However, the difference was not statistically significant, and there was no significant difference between the groups (P > .05).Finally, a total of 178 HTN patients with nonpermanent AF were followed up, including 94 males and 84 females, aged 44 to 77 years (mean 67.06 ± 8.28). There were 28 patients with diabetes, 23 patients with stroke, 74 patients with smoking history, and 51 patients with drinking history. There was no significant difference in age, gender, BMI, smoking or drinking history, diabetes, stroke and lung disease history among the four groups . At the beginning of enrollment, there was no significant difference in the inflammatory index , liver and renal function, myocardial enzyme, N‐terminal brain natriuretic peptide (NT‐proBNP) or blood lipid level among the groups (P > .05). After 24 months of treatment, the levels of hs‐CRP (mg/L) in CCB + statin group, valsartan group and valsartan + statin group were lower than those before treatment , and were significantly lower than that in CCB group (P < .05). However, no significant difference was noted in hs‐CRP between CCB + statin group, valsartan group and valsartan + statin group (P > .05). In addition, the level of NT‐proBNP (ng/L) in valsartan + statin group was significantly decreased after combined treatment . There were no significant changes in liver or renal function and myocardial enzyme level in each group compared with those before treatment, and no significant difference was observed between the groups  and LDL‐C in CCB + statin group and valsartan + statin group were obviously decreased and were significantly lower than those in CCB group and valsartan group (P > .05). But after 24 months of treatment, the measured values of LAD in CCB + statin group, valsartan group and valsartan + statin group were obviously decreased (P < .05 or P < .01). Unlike CCB group, the values of LAD and IVST in valsartan group and valsartan + statin group were significantly reduced after medication (P < .05). It is worth mentioning that the values of IVST, LVEDd and LAD in valsartan + statin group were significantly decreased after 24 months combined intervention . Although LVEF of each group was higher than before, no significant difference was observed , left ventricular end‐diastolic diameter (LVEDd), left ventricular posterior wall (LVPW), left atrial diameter (LAD) or left ventricular ejection fraction (LVEF) was found among the groups (3.4P > .05) while reexamined 7‐day holter demonstrated the f‐f interval was gradually shortened and f wave amplitude was gradually increased at 6, 12, and 24 months after corresponding treatment. In detail, reexamined holter indicated that the f‐f intervals of CCB + statin group, valsartan group and valsartan + statin group were significantly longer than that of CCB group (P < .05) while no significant difference between CCB + statin group and valsartan + statin group was noted (P > .05). However, there was no noticeable change of the f‐f interval in CCB group after drugs intervention (P > .05). Similarly, the amplitudes of f‐wave in CCB + statin group, valsartan group and valsartan + statin group were significantly higher after 24 months treatment (P < .05) while no significant difference was found in CCB group (P > .05). In addition, in CCB group, the f‐f interval moderately shortened while f wave amplitude gradually decreased during the follow‐up period. Nevertheless, in valsartan group and valsartan + statin group, their f‐f intervals extended gradually while their amplitudes of f‐wave increased slightly in the same period. Furthermore, the amplitudes of f‐wave and f‐f intervals in valsartan group and valsartan + statin group were significantly higher and longer than those in CCB group after 24 months of treatment (P < .05). Interestingly, holter suggested the f‐f interval of the 12th month in CCB group was significantly shorter than those before treatment, 6 or 24 months after treatment, therefore the specific reasons for this abnormal phenomenon need to be further studied by expanding the sample . Similarly, the mean heart rates of the four groups decreased after treatment while the trends of decline were more obvious in valsartan group and valsartan + statin group as significant decreased at the end of follow‐up  accounts for about one third of the causes of ischemic stroke. Stroke may even attack before the patient is diagnosed with AF.P < .05). In addition, after 24 months of treatment, the f‐f interval and f wave amplitude in valsartan group were longer and higher than those in the group that treated with CCB only. These results further confirmed that long‐term application of valsartan could effectively delay or even reverse left atrial enlargement and reduce atrial remodeling, improve left ventricular function, reduce sympathetic tension, enhance vagus activity, inhibit inflammation and thus reduce the incidence of AF in HTN patients.To block RAAS by ARBs has a beneficial effect in reducing the incidence of AF.P < .01); the level of hs‐CRP in valsartan group after treatment with valsartan was also decreased, but the trend in valsartan + statin group with fluvastatin was more obvious (Table P < .05). The combined treatment also improved inflammatory response and maintained heart rate variability with normal sinus rhythm. These results indirectly confirmed the role of statins in the prevention and treatment on AF.Statins, as secondary prevention drugs for coronary heart disease, can not only significantly reduce blood lipid, but also effectively decrease the incidence and mortality of cardiovascular events by regulating serum lipid levels and stabilizing artery plaque.us Table . The antIt is worth noting that in this study, we innovatively investigated the different effects of four groups drug interventions on the f‐f interval and amplitude of f‐wave. The shortening of the f‐f interval that recorded by ECG can be used as an effective index to predict the prolonged duration of paroxysmal AF and to reflect the electrical remodeling of atrium while the down regulated changes in amplitudes of f‐wave may be related to the recurrence of AF. It is apparent to find in valsartan + statin group, the trends of prolongation in f‐f interval and increase of f wave amplitude were more obvious. Compared with heart rate, which is a common index, electrocardiographic indexes f‐f interval and amplitude of f‐wave can interpret the beneficial effect of combined application of valsartan and fluvastatin on HTN patients with nonpermanent AF in a deeper level.P < .01, Table P < .05, Table As mentioned above, existing interventions cannot completely cure AF, and paroxysmal AF always progresses to permanent AF. Our results were consistent with the above consensus. After 24 months of follow‐up, the heart rate of the four groups decreased, but the AF load increased significantly. However, the AF load in valsartan group and valsartan + statin group was significantly lower than that in CCB group after treatment (all There are also some limitations in this study. First, although this study is a multicenter clinical study, there are few selected cases, and the follow‐up time is short, which affects the persuasion of the test results. Further larger randomized clinical trial will be necessary to confirm the conclusions of our study. Secondly, this study did not assess the effect of application of fluvastatin alone on AF, and failed to directly prove whether fluvastatin can prevent AF independently. At last, although 7‐day Holter can capture most AF episodes, the potential subclinical AF may be underestimated.To sum up, our study is a multicenter, randomized, open, four group parallel design clinical trial to investigate whether valsartan and its combination with fluvastatin can reduce the recurrence of paroxysmal AF in patients with HTN and nonpermanent AF and delay the progression of nonpermanent AF to permanent AF compared with CCB or CCB combined with fluvastatin. Our results suggest that valsartan can decrease the occurrence of AF by reducing myocardial load, inhibiting atrial remodeling, exerting anti‐inflammation, and regulating nerve tension. Similarly, fluvastatin has the effects of anti‐inflammatory and improving atrial remodeling. The combination of valsartan and fluvastatin can significantly improve heart function, reduce the AF load, maintain normal sinus rhythm, and delay the time of nonpermanent AF to permanent AF. It is obvious that the combination of the two drugs can produce synergistic therapeutic impact and improve the therapeutic effect. RAAS blocker combined with statins is a highlight of nontraditional antiarrhythmic drugs in the treatment of cardiac arrhythmia. With further validation of basic research and large‐scale clinical trial, RAAS blocker combined with statins may play a certain role in the prevention and treatment of AF, which is expected to become a new strategy for AF upstream control.The authors declare that there is no conflict of interest.The data used to support the findings of this study are available from the corresponding author upon request.


Several hematological indices including subtypes of leukocytes populations have been associated with cardiovascular outcome. Takotsubo syndrome (TTS) is a form of acute heart failure syndrome featured by several in‐hospital complications (IHCs).Hematological indices at admission may predict IHCs in TTS patients.One hundred and sixty consecutive patients with TTS were enrolled in a multicenter prospective registry. Clinical data, admission hemogram, and IHCs were recorded.Incidence of IHCs was 37%, including pulmonary edema 9%, cardiogenic shock 9%, need of invasive ventilation 10%, death 8%, stroke 2.5%, and left ventricular thrombi 6%.P = .002) and white blood cells/mean platelet volume ratio  at admission were significantly higher in patients with IHCs.Patients with IHCs were older, more frequently male, with physical stressor‐induced TTS, lower left ventricular ejection fraction at admission. Neutrophil/lymphocyte ratio (NLr) . When stratified according to NLr into tertiles, the rate of IHCs was from first to third tertile was, respectively, 22%, 31%, and 58%. NLr values in the higher tertile were independent predictors of IHCs even at multivariate analysis .NLr values were predictor of IHCs .NLr is an independent predictor of IHCs in patients admitted with TTS. Admission hemogram may represent a potential tool for prediction of IHCs in TTS. The entire study population fulfilled the 2008 revised Mayo Clinic diagnostic criteria: (a) transient hypokinesis, dyskinesis, or akinesis of the left ventricular (LV) apical and/or midventricular or basal segments extending beyond a single epicardial vessel distribution territory; (b) absence of significant obstructive coronary artery disease explaining the extent of wall motion abnormalities or angiographic evidence of acute plaque rupture; (c) new electrocardiogram (ECG) abnormalities (either ST‐segment elevation and/or T‐wave inversion) or modest elevation in the cardiac troponin levels; (d) absence of pheochromocytoma or myocarditis or intracranial hemorrhage.The study was held according to declaration of Helsinki and approved by local ethics committees; all participants provided a written informed consent.2.2All patients underwent a clinical examination and a detailed anamnesis was collected including: age, gender, and kind of triggering stressor. Medical history, including history of neurologic disorders , was also recorded.A two‐dimensional Doppler Echocardiographic examination at admission, at third day, and at discharge was performed. The left ventricular ejection fraction (LVEF) was calculated using the Simpson method from the apical four‐chamber and two‐chamber view. Coronary angiography was performed in all patients at admission.2.39/L for the platelet count, 4.3 to 10.0 × 109/L for total white blood cells, 2.0 to 7.0 × 109/L for neutrophils, and 0.95 to 4.5 × 109/L for lymphocytes. The entire investigation was carried out using the same analyzer and the quality of results was validated throughout the study period by regular internal quality control procedures and participation in an external quality assessment scheme.The complete blood cell count was performed after blood samples with K2EDTA tubes , which were always collected at the time of admission. The analysis was performed with the fully automated hematological analyzer Advia 2120 . The local reference ranges are 150 to 400 × 10The values of NLr, white blood cell count/mean platelet volume (MPV) ratio, MPV/platelets ratio, and platelet count/lymphocyte ratio were, respectively, calculated by dividing the absolute number of each variable obtained in the same blood sample collected at admission.2.4The primary clinical endpoint was occurrence of IHCs including overall mortality, pulmonary oedema, need of invasive ventilation, cardiogenic shock, stroke, and LV thrombosis.2.5t test or Mann‐Whitney U test as required. Categorical variables were presented as percentages and compared with χ2 or Fisher test as required. The Kolmogorov‐Smirnov test was used to identify variables with normal distribution.Continuous variables were expressed as mean ± SD and compared with Student Logistic regression analysis was used to identify predictors of outcome. Forward multivariable logistic regression analysis was used for correcting for principal confounders. Predictors at univariable logistic regression with a significance value ≤0.01 were included in multivariable analysis. Odds ratios (OR) with 95% confidence interval (CI) were calculated. Linear correlations were determined by measuring the Pearson's correlation coefficient.P < .05 was considered as statistically significant.A 33.1Mean age was 74 ± 12 years, mean LVEF 36 ± 8%, 11% (17 patients) were male (Table P < .01), more frequently male , more frequently presented a physical stressor , with higher prevalence of infective/inflammatory triggers (26 vs 12% P = .03) or absence of chest pain , and a lower LVEF at admission  , neutrophils , and lower values of eosinophils (0.03 ± 0.1vs 0.08 ± 0.1 103/μL P = <.01). NLr  and white blood cells/MPV ratio  were significantly higher in patients with IHCs  and significantly correlated with hospital stay duration    Figure . NLr valP < .01)  Patients that experienced IHCs presented higher values of white blood cells and neutrophils at admission;2) NLr is an independent predictor of IHCs;3) When stratified according to NLr tertiles, values in the higher tertile were independent predictors of IHCs.TTS has been for a long time considered a benign disease, because of the completely reversible nature of the distinct wall motion abnormalities. However, several clinical studies have shown that TTS is a form of acute heart failure that can be associated with life‐threatening complications.The role of hemogram parameters in cardiovascular diseases has been extensively studied and a relationship between several inflammatory markers and cardiovascular diseases has been established mainly in the context of coronary artery disease.Neutrophils represent the first type of white cells that is generally observed, following damage, in myocardial tissue.High levels of white blood cells have been associated with an increased risk of mortality in patients with ACS.Platelets levels in term of platelet counts and MPV may be higher in ACS as a result and a precipitating factor of inflammatory response. Megakaryocyte could be stimulated by several inflammatory mediators and present accelerated proliferation and platelet‐production. On the other hand, platelets can release mediators that promote the progression of atherosclerosisA systemic inflammatory activation can be observed in TTS as well as in ACS. We previously found that, in the acute and sub‐acute phase of TTS, serum levels of anti‐inflammatory interleukins  are higher when compared with ACS patients,Murakami et al,When compared to other parameters, NLr predicted adverse events in ACS. In a meta‐analysis on 10 245 patients with ST‐elevation acute myocardial infarction after percutaneous angioplasty, Zhang et al The question whether lymphocyte anomalies are induced by catecholamine surge or myocardial dysfunction, however, still remains unresolved, deserving further future investigations.5These are preliminary data to be confirmed in larger cohorts of patients. Further and more adequately powered prospective studies are warranted to clarify the assay standardization, the optimal cutoff, and the prognostic value of NLr in association with other biomarkers and clinical scores.Prior to admission hemograms were unknown and a comparison with admission hemograms was not possible; baseline conditions associated with immunomodulation or chronic leukocytosis were not ruled out.6NLr is an independent predictor of IHCs in patients admitted with TTS. Admission hemogram may be an easy and fast tool for risk stratification of IHCs in TTS.The authors declare no potential conflict of interest.All authors have read and approved the manuscript.


Climatic and dietary shifts predispose ruminal microbes to hyperthermal and hyperosmotic stress, leading to poor fermentation and subsequently adverse effects on ruminant productivity. Betaine may function as substrate, osmolyte, antioxidant, and methyl donor for microbes. However, its effect depends on the extent of microbial catabolism. This study revealed the ruminal disappearance kinetics of betaine and its dose effect on ruminal fermentation during thermal and osmotic stress using a rumen simulation technique.−1; each was assigned to two incubation temperatures (39.5 and 42 °C) and two osmotic conditions (295 and 420 mOsmol kg−1). Betaine disappeared rapidly within the first 6 h of incubation; however, the rate was lower during hyperosmotic stress (P < 0.05), the stress condition that also suppressed the overall fermentation and degradation of organic nutrients and decreased the bacterial diversity (P < 0.001). During hyperosmotic stress, betaine shifted the fermentation pathway to more propionate (P < 0.05). Betaine counteracted the negative effect of hyperthermal stress on total short‐chain fatty acid concentration (P < 0.05) without affecting the composition. Both stress conditions shifted the bacterial composition, but the effect of betaine was minimal.Three different betaine doses were used: 0, 50, and 286 mg LJournal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.Despite its rapid ruminal disappearance, betaine modulated microbial fermentation in different ways depending on stress conditions, indicating the plasticity of the betaine effect in response to various kinds of physicochemical stress. Although betaine did not affect the abundance of ruminal microbiota, the enhanced fermentation suggests an improved microbial metabolic activity under stress conditions. © 2020 The Authors.  Ruminants are dependent upon ruminal microbes, which enable many biochemical functions, such as formation of short‐chain fatty acids (SCFAs) and microbial protein synthesis by fermenting feedstuffs that are typically not digested by the host.Betaine, or trimethylglycine, is found naturally in microbes, animals, and plants, and it is highly abundant in sugar beet.To fill this research gap, this study was designed with the goal to investigate ruminal disappearance kinetics of betaine and the dose‐dependent effects on rumen microbial composition and ruminal fermentation characteristics under hyperthermal and hyperosmotic conditions, using a rumen simulation technique (Rusitec). We hypothesized that increased temperature and hyperosmolality would reduce microbial fermentation and population diversity. However, more betaine would be utilized by ruminal microbes to maintain fermentation and diversity under stress conditions, and thus supplementing betaine could beneficially modulate rumen fermentation.n = 6 per individual treatment. Each experimental run lasted for 10 days, with the first 5 days specified as an adjustment for equilibration of the system followed by the last 5 days for sampling. The test conditions were arranged as a 2 × 2 × 3 factorial design including two temperatures , two osmotic conditions , and three target betaine concentrations in the incubation liquid at 0 (CON), 51 (LB) and 286 mg L−1 (HB). The betaine doses were prepared from betaine obtained from sugar beet molasses . Target temperatures were maintained by thermostatically controlled water baths. The high temperature chosen (42.0 °C) was based on the maximum temperature observed in cattle exposed to mild heat stress conditions.in vivo in response to feeding.The experiment was carried out using two Rusitec systems, forming a total of 12 experimental units used in each experimental run. The trial was performed with six experimental runs, resulting in On the first day of each experimental run, the ruminal fluid and solid digesta were collected from three fistulated non‐lactating Holstein cows kept at the dairy research station (VetFarm) of Vetmeduni, Vienna. The donor cows were fed primarily hay and grass silage and cared for according to the Austrian guidelines of animal welfare.For daily feed bag exchange, the feed bag incubated for 48 h was removed and replaced with a fresh feed bag. Before removal, the incubated feed bag was rinsed and squeezed with 40 mL of the pre‐warmed respective buffer. The effluent bottle was emptied and the gas‐tight bag changed daily. After feed bag exchange, the fermenter was closed and flushed with nitrogen gas for 3 min to re‐establish anaerobic conditions. Shortly afterward, betaine was thoroughly dosed into the fermenter via the valve.During the sampling period (d6–d10) the 48 h incubated feed bags were washed with cold water in a tub until the water became clear and then stored at −20 °C for later analysis of dry matter, organic matter (OM), crude protein, crude fat, and neutral detergent fiber (NDF) according to the method of VDLUFA.g for 15 min. The samples were then filtered with a 0.45 μm filter and the clear solution was injected into high‐performance liquid chromatograph without further dilution. Separation of the betaine was done on a Bio‐Rad Aminex HPX 87‐K column. The injection volume was kept at 20 μL, and the oven temperature was fixed at 65 °C. Afterward, the separation was performed under isocratic elution conditions using a mobile phase containing 50 mmol L−1 dipotassium hydrogenphosphate and 50 mmol L−1 monopotassium phosphate. The mobile phase was delivered at a flow rate of 0.6 mL min−1, and quantification was based on refractive index detection.The fermenter fluid was collected daily (d6–d10) from each fermenter to measure ruminal fermentation characteristics as described previously.−1 lysozyme; 10 μL of 2.5 U mL−1 mutanolysin , 21 μL of 18.6 mg mL−1 proteinase K (Sigma Aldrich) and mechanical disruption ) to improve DNA efficiency, according to previously published procedures.et al.On d10, fermenter fluid was collected and snap‐frozen in liquid nitrogen, and solid samples were collected and all samples stored at −20 °C for microbial analysis. Total microbial DNA was extracted from about 800 μL of fluid and 0.25 g of solid digesta using the DNeasy PowerSoil Kit  following the method described previously,a and b). Subsequently, each parameter was statistically analyzed using the MIXED procedure of SAS for the same main effects in the model as mentioned earlier. Correlation analysis was performed using the CORR procedure to obtain Pearson correlation coefficients. Mean values reported are least square means plus/minus the pooled standard error of the mean (SEM). Significance was declared at P ≤ 0.05 and a tendency of an effect at 0.05 < P ≤ 0.10.Statistical analyses were performed using the MIXED procedure of SAS . The statistical model included betaine supplementation, incubation temperature, and osmolality along with their two‐way and three‐way interactions. The variation between experimental runs was considered as a random effect, and data for the daily measurements of the same fermenter were analyzed as a repeated measure with compound symmetry as the variance–covariance structure. Only for betaine added groups was the disappearance kinetics of betaine fitted as an exponential decay function (−1 (mean plus/minus standard deviation) for LB and 314 ± 41 mg L−1 for HB. The 0 h concentration of betaine in the CON group was 0.8 ± 1.2 mg L−1.As analyzed, the concentration of betaine in the incubation liquid at 0 h was 56 ± 6 mg Lb was found with LB under the physiological condition, whereas the lowest b was found with HB with the hyperosmotic condition (P < 0.05). The HB group incubated under the hyperosmotic condition maintained a higher betaine concentration than the other groups did (P < 0.05) during the first 6 h after addition. No betaine could be detected at 24 h after addition.Betaine rapidly disappeared in the first 6 h after addition; however, the disappearance rate was dose dependent and was affected by the osmolality of fermenter fluid, but not by the incubation temperature Fig. , Table 3P < 0.05). Total SCFAs concentration per OM degraded was higher in LB and HB than in CON (P < 0.05).Table P = 0.087), acetate concentration (P = 0.067), and the molar percentage of isovalerate (0.022) (Table P < 0.05), whereas HB showed intermediate values. Without betaine, the hyperthermal condition decreased concentrations of total SCFAs (P < 0.10) and propionate (P < 0.05), but LB and HB reversed the effect of hyperthermal condition on total SCFAs.Interaction between betaine addition and incubation temperature tended to be significant for total SCFAs concentration 2) Table . As show Table . P < 0.01). There was an osmolality × betaine interaction for molar percentages of acetate (P = 0.047) and propionate (P = 0.076) and acetate to propionate ratio (P = 0.045) (Table P < 0.05). Both LB and HB increased acetate percentage compared with CON (P < 0.05) under the physiological condition and promoted propionate percentage under the hyperosmotic stress (P < 0.05).Overall, the hyperosmotic condition decreased overall fermentation, as seen by lowered SCFAs concentration compared with those under the physiological condition (e P = 0.07 and pro5) Table . WithoutP < 0.05). The hyperthermal condition increased the disappearance of NDF at the expense of NFCs disappearance (P < 0.05).Betaine addition did not affect the nutrient disappearances, except for a tendency for betaine × osmolality interaction for crude protein and fat disappearances Table . The hypP < 0.001), whereas temperature and betaine addition did not affect the diversity.All microbial diversity indices including Chao1, Shannon, and Simpson indices were lower in the hyperosmotic condition than in physiological conditions , with the exception of Firmicutes and Cyanobacteria (P ≤ 0.10). The hyperosmotic condition increased the relative abundance of the three dominant phyla, and Proteobacteria, whereas it decreased the abundance of the other minor phyla. The hyperthermal condition decreased the relative abundance of the phyla Fibrobacteres, Synergistetes, TM7, and Verrucomicrobia (P < 0.05). Of the identified phyla, betaine affected the relative abundance of Chloroflexi depending on the osmolality.yla Fig. . OsmolalP ≤ 0.05), whereas betaine affected only three genera of low abundances, including CF231, SHD231, and Mogibacterium . Prevotella, Lactobacillus, and Bifidobacterium, which were the microbial genera with high abundances, increased under the hyperosmotic condition. Nineteen minor genera were sensitive and nine other minor genera were resistant to the hyperosmolality. The hyperthermal condition increased the relative abundance of Butyrivibrio, Pseudoramibacter_Eubacterium, and Succiniclasticum but decreased Succinivibrio and CF231.The relative abundance of microbial genera was mainly affected by osmolality and temperature P ≤ 0.0, whereasThe addition of betaine in the rumen could be of high significance to the animal because betaine, as a substrate and methyl donor for microbes, directly modulates ruminal fermentation14C]‑ and [methyl‐14C]‐labeled betaine, Mitchell et al.in vivo. Much slower disappearance rates were found in studies using a batch culture technique and testing very high betaine doses (>500 mg L−1).in vitro liquid, another source of betaine disappearance could be attributed to the incorporation by microbial cells in order to maintain water and ionic balances.In the present Rusitec experiment, we observed a rapid disappearance of betaine. Under physiological conditions in terms of osmolality and pH, about 5–15% remained at 4 h, with more betaine being recovered when administered at the high dose. By using [1‐et al.,We showed for the first time that the extensive disappearance of rumen‐unprotected betaine could be delayed by hyperosmolality, whereas hyperthermal stress had no effect. The findings, however, did not reflect our hypothesis that more betaine would be incorporated into microbial cells, which will enable them to adapt themselves to fluctuations in external osmotic pressures.in vitro data showed that the hyperthermal stress suppressed ruminal fermentation, which was mainly attributed to decreased propionate production. This explains previous indications that heat stress alters carbohydrate metabolism, leading to lower plasma glucose and lactose secretion in the milk.Succinivibrio.Fibrobactor, which was consistent with a previous study in cattle.Ruminococcus,−1 more milk production in mid‐lactation Holstein cows when betaine was supplemented in the diet during hot months of the year.Betaine acts as a thermoprotectant−1, resembling the postprandial range in vivo,et al.−1. In our study, the major fibrolytic bacteria  were sensitive to hyperosmolality, whereas amylolytic bacteria, propionate producers, and acid‐tolerant bacteria  proliferated, a characteristic that has been previously associated with high‐grain feeding.The presence of betaine could not reverse the negative effect of hyperosmolality on suppressing fermentation; instead, betaine shifted the fermentation pathways. The increased molar percentage of acetate with betaine addition was evident under normal osmolality. With elevated osmolalities above 400 mOsmol kgin vitro, but the degradation was delayed during hyperosmotic stress, the stress condition that also largely compromised overall degradation of major nutrients and shifted the fermentation pathway to more propionate. Without affecting the microbial composition and diversity, betaine supplementation altered their functionality and, therefore, promoted ruminal fermentation manifested by the dominant microbial community. In a nutshell, ruminal microbiota can genuinely benefit from betaine to support their metabolism and growth.Betaine was rapidly degraded by ruminal microbiota


To explore epidemiological features of demodicosis relevant to UK veterinary general practitioners. Breed risk factors were proposed as distinct between juvenile‐onset and adult‐onset disease.The study used anonymised clinical data on dogs under primary veterinary care at practices enrolled in the UK VetCompass Programme. Case inclusion required recording of a final demodicosis diagnosis for a dermatological condition that was present during the 2013 study period. Risk factor analysis used multivariable logistic regression modelling.In dogs aged <2 years (juvenile‐onset), the 1‐year period prevalence was 0.48% . Compared with crossbred dogs, seven breeds showed increased odds of diagnosis with demodex: British bulldog, Staffordshire bull terrier, Chinese shar‐pei, dogue de Bordeaux, pug, French bulldog and boxer. Additionally, six breeds showed reduced odds of juvenile demodicosis: Lhasa apso, bichon frise, Labrador retriever, German shepherd dog, shih‐tzu and Chihuahua. In dogs aged >4 years (adult‐onset), the 1‐year period prevalence was 0.05% . Six breeds showed increased odds of demodicosis compared with crossbred dogs: Chinese shar‐pei, shih‐tzu, West Highland white terrier, pug, boxer and Border terrier.Juvenile‐onset demodicosis is much more common (about 10 times higher) than the adult‐onset form. Knowledge of the predisposed breeds for these two presentations can assist with diagnosis and support the concept of distinct aetiopathogenetic phenotypes. Demodex canis, was first recognised in 1859  and/or the extent of the disease  to assist with prognostic advice and to guide diagnostic and management approaches  to report the 1‐year period prevalence and age distribution of demodicosis in dogs of all ages attending primary care practices in the UK; and, (2) to report the 1‐year period prevalence and evaluate risk factors for demodicosis separately for dogs under 2 years of age (juvenile‐onset) and for those aged over 4 years (presumed adult‐onset). The study proposed that breed risk factors are distinct between the juvenile and adult‐onset forms of the disease.et al. et al. The VetCompass Programme collates de‐identified electronic patient record (EPR) data from primary‐care veterinary practices in the UK for epidemiological research  or ongoing cases first diagnosed before 2013 (pre‐existing cases). Case‐finding involved initial screening of all 455,553 study dogs for candidate demodicosis cases by searching the clinical free‐text field using the search term “demod*” with fuzziness to allow two‐letter inversion, insertion or deletion  and a grouped category of all remaining dogs. This approach was taken to facilitate statistical power for the individual breed analyses . Adult bodyweight described the maximum bodyweight (kg) recorded for dogs >18 months at any time in the available clinical records. An adult bodyweight variable categorised adult bodyweight into six groups . A bodyweight relative to breed/sex mean variable characterised the adult bodyweight of individual dogs as either below or equal/above the mean adult bodyweight for their breed type and sex within the overall study population. Crossbred was included as a single breed type for this variable. This variable allowed the effect of adult bodyweight to be assessed within each breed/sex combination.Breed information recorded in the clinical records was mapped to a standardised breed list adapted from the VeNom Coding system (The VeNom Coding Group e.g. 1 year) and is generally reported as the percentage of cases from all animals that were at risk. Prevalence describes all cases, whether incident or pre‐existing  and demodicosis during 2013. Based on review of the current literature on reported risk factors and pathogenesis of demodicosis within possible causal pathways  and adult bodyweight  were not considered in the same multivariable modelling. Instead, purebred replaced the breed variable in the final breed‐multivariable while adult bodyweight replaced the breed and bodyweight relative to breed/sex mean variables. Model development used manual backwards stepwise elimination. The likelihood ratio test was used to compare a random effects model with clinic entered as a random effect against the non‐random effects model with P<0.05 cut‐off used for selection of the random effects model. Pair‐wise interaction effects were evaluated using the likelihood ratio test for all variables retained in the final models, with P<0.05 cut‐off used for inclusion of biologically plausible interaction terms , analyses were conducted using Stata Version 13 (Stata Corporation). Period prevalence refers to the number of cases that are known to have existed at any time during a specified period . The breeds with the highest demodicosis 1‐year period prevalence across all ages were British bulldog, French bulldog, pug, dogue de Bordeaux and Chinese shar‐pei . The breeds with the highest demodicosis 1‐year period prevalence among dogs aged <2 years were British bulldog, Staffordshire bull terrier, pug, French bulldog, Chinese shar‐pei and dogue de Bordeaux . The breeds with the highest demodicosis 1‐year period prevalence among dogs aged >4 years were Chinese shar‐pei, shih‐tzu, West Highland white terrier, dogue de Bordeaux and pug . The intraclass correlation coefficient indicated that 5.9% of the unaccounted‐for variation in the data was due to unmeasured factors operating at the veterinary clinic level. No biologically plausible interactions at P<0.05 were detected. The final unclustered model showed acceptable model‐fit (Hosmer‐Lemeshow test statistic: P=0.311) and good discrimination (area under the ROC curve: 0.841). The frequencies of missing data for the 126,423 dogs in the final model variable were: breed n=492; 0.4%, bodyweight relative to breed/sex mean n=75,878; 60.0%, age n=0; 0.0% and sex‐neuter n=16,901; 13.4%.Six risk factors had univariable association with demodicosis at P<0.20 and were considered in multivariable modelling: After accounting for the effects of the other variables evaluated, seven breeds showed increased odds of demodicosis in dogs aged <2 years compared with crossbred dogs: British bulldog , Staffordshire bull terrier , Chinese shar‐pei , dogue de Bordeaux , pug , French bulldog  and boxer . There were six breeds with reduced odds of demodicosis compared with crossbreds: Lhasa apso, bichon frise, Labrador retriever, German shepherd dog, shih‐tzu and Chihuahua. Additionally, zero cases were identified for Yorkshire terrier and golden retriever. Dogs with an adult bodyweight that was equal or higher than their breed mean had 1.50  times the odds of demodicosis compared with dogs that weighed below their breed mean. Dogs aged 1.0 to <2.0 years had 0.12  times the odds of demodicosis compared with dogs aged <1.0 years. Compared with entire females, there were higher odds in neutered females  and neutered males  (Table purebred and adult bodyweight) were individually added to the final breed‐multivariable model, replacing the breed variable. Purebred dogs had 1.50 times the odds  compared with crossbred dogs. Dogs weighing 10.0 to 19.9 kg  and 20.0 to 29.9 kg  had the highest odds of demodicosis compared with dogs weighing <10.0 kg , shih‐tzu , West Highland white terrier , pug , boxer  and Border terrier . There were four breeds with zero demodicosis cases identified: French bulldog, Border collie, Chihuahua and bichon frise. Purebred dogs had 2.16 times the odds  compared with crossbred dogs. Dogs weighing 20.0 to 29.9 kg  and 30.0 to 39.9 kg  had lower odds of demodicosis compared with dogs weighing <10.0 kg , and adult‐onset demodicosis, usually first manifesting after 4 years of age  and/or an acquired mite‐specific immune dysfunction.Three breeds, shih‐tzu, West Highland white terrier and Border terrier, had increased odds only for dogs aged >4 years. West Highland white terrier and shih‐tzu were previously reported as the most frequently affected purebred dogs among a population of dogs with adult‐onset demodicosis in a teaching hospital (Bowden e.g. obesity) or simply larger inherent stature/size. The current study did not have access to accompanying morphometric data (e.g. body condition) on individual dogs and therefore it was not possible to interpret the causes of differing bodyweights  odds for demodicosis. Heavier bodyweight could reflect either increased body condition  than in dogs aged over 4 years (0.05%). It identified breed as a major risk factor for both age groups. Differing spectra of predisposed breeds across the juvenile‐onset and adult‐onset forms of the disease, and in particular the dominance of bull type breeds in clade W from the 23 identified genetic clades (Parker All authors made substantial contributions to conception and design, acquisition and extraction of data, and to analysis and interpretation of the results. All authors were involved in drafting and revising the manuscript and gave final approval of the version to be published. Each author agrees to be accountable for all aspects of the accuracy or integrity of the work.The authors have no conflicts of interest to declare.


Asymmetric dimethylarginine (ADMA) has been proposed as a severity marker in humans with acute pancreatitis (AP).To evaluate ADMA in dogs with AP compared with healthy dogs and its association with severity of disease, mortality, and indicators of the systemic inflammatory response syndrome (SIRS), including serum C‐reactive protein (CRP) concentration, WBC count, and band neutrophils.Fifty‐four dogs with AP and a control group (CG) of 28 healthy dogs.Cohort study including dogs with AP diagnosed using clinical and laboratory variables, abnormal canine pancreatic lipase (cPL) concentration, and compatible abdominal ultrasound examination findings performed within 48 hours of admission. Canine AP severity (CAPS) was calculated. Serum concentration of ADMA was measured using high performance liquid chromatography. Blood donor‐, staff‐, and student‐owned dogs were enrolled in the CG.P = .003). Dogs with CAPS ≥11 had higher serum ADMA concentrations than did dogs with CAPS <11 (92 versus 54.6 μg/dL P = .009). Univariable analysis for mortality, CAPS score, band neutrophils, CRP, and ADMA were included in multivariable logistic regression, in which only ADMA was associated with mortality (P = .02). Survivors had a significant decrease in ADMA at first reevaluation compared to admission (P = .02).Dogs with AP had higher median admission serum ADMA concentrations compared with the CG (62 versus 48.5 μg/dL; Because serum ADMA concentrations were higher in AP dogs compared with the CG, it may have value as a biomarker in the diagnosis of AP in dogs. In addition, because ADMA was associated with mortality, it may have prognostic value. ADMAasymmetric dimethylarginineAPacute pancreatitisCAPScanine acute pancreatitis severityCGcontrol groupcPLcanine pancreatic lipaseCRPC‐reactive proteinNOnitric oxideNOSnitric oxide speciesSIRSsystemic inflammatory response syndrome1Acute pancreatitis (AP) is the most common disease of the exocrine pancreas in dogs.To the best of our knowledge, no studies in veterinary medicine have evaluated the relationship between ADMA and AP. However, ADMA has been studied in SIRS and sepsis in humansOur hypothesis was that ADMA would be a marker of disease severity and inflammation in dogs with AP. Our aims were to evaluate serum ADMA concentrations in dogs with AP compared with a control group of healthy dogs (CG) and assess possible relationships between ADMA and severity of disease, acute inflammatory markers  and survival in dogs with AP.2Serum ADMA concentrations were evaluated using surplus serum of hospitalized dogs with AP included in a larger prospective study conducted at our veterinary teaching hospital between March 2017 and April 2019, for which ethical approval had been obtained . Surplus serum samples from each dog were frozen at −80°C at the time of sampling  pending analysis, which was performed ≤25 months after collection.A diagnosis of AP was made if all of the following criteria were met: compatible clinical signs , changes consistent with AP on abdominal ultrasound examination  for AP within 48 hours of hospital admission and abnormal SNAP test result for canine pancreatic lipase (cPL) . Abdominal ultrasound examination was performed by an experienced radiologist and was considered consistent with AP if the pancreas was hypoechoic and enlarged, with irregular shape and margins, and surrounded by hyperechoic mesentery or abdominal effusion or both.In our facility, serum samples from blood donors, and staff, or student‐owned healthy dogs are routinely stored at −80°C for scientific purposes and each owner signs informed consent for such usage. Dogs were considered healthy based on history, physical examination, and blood test results . Twenty‐eight healthy dog serum samples were available for the CG for ADMA analysis.Each AP dog had a CBC, serum biochemical profile, coagulation profile, urinalysis, and venous blood gas analysis performed as part of routine care for AP. Data from CBC as well as serum creatinine and ionized calcium concentrations at presentation were recorded.Canine acute pancreatitis severity (CAPS), a recently validated clinical scoring system for short‐term mortality in dogs with AP,The CAPS score was calculated as follows: CAPS score = 8 ×  + 3 ×  + 4 ×  + 3 × .Mortality was evaluated at hospital discharge and dogs were divided into survivors  and nonsurvivors .Approximately 2 weeks (10‐20 days) after discharge, survivors were clinically reevaluated. A CBC and serum biochemical profile were performed, and surplus serum was stored at −80°C until analysis.Serum ADMA concentrations were measured both at T0  and at the first reevaluation (T1). Serum concentrations of ADMA were determined using high performance liquid chromatography with fluorescence detection, as previously described.2.1All continuous variables were tested using the Kolmogorov‐Smirnov normality test. Normally distributed variables were expressed using the mean ± SD, and nonnormally distributed variables were expressed using the median and range.U test.The T0 serum ADMA concentration was compared between AP dogs and CG using a Mann‐Whitney U test was performed to determine if T0 ADMA was different between dogs with and without SIRS and between CAPS score groups (< or ≥11). Spearman's or Pearson's correlation test was performed between ADMA and serum C‐reactive protein (CRP), WBC, and band neutrophil count.A Mann‐Whitney U test based on data distribution  or chi‐square test (CAPS score groups and SIRS). All variables with a P‐value <.2 were subjected to multivariable analysis. Afterward, multivariable backward stepwise binary logistic regression was performed to assess the association of variables with mortality, using the variables previously identified in the univariable model as significantly associated with mortality. If a variable remained significantly associated with mortality (P < .05), it was considered to be associated with the death of the dog.A univariable analysis was performed to identify variables associated with mortality using t‐tests or the Mann‐Whitney t test) to establish if, after remission of AP, survivors had undergone a significant change in serum ADMA concentration.Finally, T1 serum ADMA concentrations in survivors were compared with T0 ADMA .33.1Fifty‐four dogs diagnosed with AP were enrolled with the owners’ informed consent. Mean age was 10.4 ± 3.5 years. The most commonly affected breeds were Poodle (n = 4), German Shepherd (n = 2), Lagotto Romagnolo (n = 2), Labrador Retriever (n = 2), Beagle (n = 2), Jack Russell terrier (n = 2), Springer Spaniel (n = 2), Shih Tzu (n = 2), and Spitz (n = 2). Seventeen dogs (33%) were mixed breed and the remaining 17 dogs belonged to other breeds . Twenty‐five of 54 dogs (46.3%) were females, of which 8 were spayed, whereas the other 29 dogs were males (53.7%), of which 12 were castrated. The median weight was 16.5 kg, ranging from 3.27 to 40.7 kg. In our cohort of dogs, the CAPS score ranged from 0 to 15 with a median of 8. One dog had a CAPS score of 3, 31 dogs had CAPS scores of 8, whereas 5 and 8 dogs had CAPS scores of 11 and 12, respectively. Seventeen dogs (31.5%) were in the nonsurvivor group. The CG included 28 mixed breed dogs 4.5 ± 4 years of age. Mean weight of the CG was 26 ± 19 kg.3.2P = .003; Figure P = .009; Figure P = .24). In addition, no significant correlation was found between serum ADMA concentration and CRP, WBC, or band neutrophil count .The AP dogs had significantly higher median serum ADMA concentration compared with the CG .Sixteen of 37 serum samples (43.2%) were available for determination of T1 serum ADMA concentration. All of the survivors had resolution of the clinical signs of AP and a significant decrease in T1 compared with T0 serum ADMA concentrations , but it was not associated with the presence of SIRS or correlated with inflammatory markers commonly used in clinical practice (CRP and WBC). Furthermore, serum ADMA concentrations were significantly lower in the survivor group, after resolution of AP.Serum ADMA concentration already has been reported as a marker of endothelial dysfunction in dogs, and nonsurvivors may have more severe oxidative stress, which may lead to an increase in serum ADMA concentration. However, we failed to find any association between serum ADMA concentration and SIRS, CRP, or WBC. This perhaps was because serum ADMA concentration appeared to be more related to oxidative stress status than to SIRS itself.Serum ADMA concentration was studied primarily in septic human patients, in which it recently was identified as a prognostic marker.Our study had several limitations. In the follow‐up analysis, serum ADMA concentrations were measured in only 16 dogs of the survivor group, and it would have been valuable to have a larger study population. Secondly, other measurements of oxidative stress, such as NOS, reactive oxygen metabolites, and biological antioxidant potential could have provided more information on the oxidative stress status in our dogs and, thus, a better understanding of the role of serum ADMA concentration in dogs with AP. Thirdly, we chose to evaluate serum ADMA concentration in relation to SIRS and other widely used inflammatory markers as WBC, CRP and band neutrophils, however, it could be interesting to study ADMA in relation to other markers, such as cytokines. Breed, age, and sex have not been explored in relation to serum ADMA concentration. Finally, naturally occurring AP in dogs is very often accompanied by or caused by other conditions. However, such comorbidities could not necessarily be ruled out, and they were not taken into account in evaluating mortality.In conclusion, our study suggests an association between high serum ADMA concentration and death in dogs with AP. Finally, as reported in humans, in our study serum ADMA concentration was significantly decreased over time in dogs that survived.Authors declare no conflict of interest.Authors declare no off‐label use of antimicrobials.The study was approved by the Scientific Ethical Committee for Animal Testing of the University of Pisa Approval No. 16749/2017.Authors declare human ethics approval was not needed for this study.


The increased availability of patient reported outcome data makes it feasible to provide patients tailored risk information of cancer treatment side effects. However, it is unclear how such information influences patients’ risk interpretations compared to generic population-based risks, and which message format should be used to communicate such individualized statistics.n = 141) read a hypothetical treatment decision-making scenario about four side effect risks of adjuvant chemotherapy for advanced colon cancer. Participants were cancer patients or survivors who were recruited from an online Dutch cancer patient panel. All participants received two tailored risks  and two generic risks conveying the likelihood of experiencing the side effects. The risks were presented either in words-only (‘common’ and ‘very common’), or in a combination of words and corresponding numerical estimates . Participants’ estimation of the probability, accuracy of their estimation, and perceived likelihood of occurrence were primary outcomes. Perceived personal relevance and perceived uncertainty were secondary outcomes.A web-based experiment was conducted in which participants (Tailored risks were estimated as higher and less accurate than generic risks, but only when they were presented in words; Such differences were not found in the verbal and numerical combined condition. Although tailoring risks did not impact participants’ perceived likelihood of occurrence, tailored risks were perceived as more personally relevant than generic risks in both message formats. Finally, tailored risks were perceived as less uncertain than generic risks, but only in the verbal-only condition.how tailored risks of treatment side effects should be communicated to patients. We recommend both clinicians who communicate probability information during consultations, and decision aid developers, that verbal descriptors of tailored risks should be supported by numerical estimates of risks levels, to avoid overestimation of risks.Considering current interest in the use of personalized decision aids for improving shared decision-making in oncology, it is important that clinicians consider  After a cancer diagnosis, most patients want to be fully informed about the possible treatment options and the associated risks of side effects to support a well-informed treatment decision-making process , 2. For Tailoring risk information of side effects adjusted to the clinical  and sociodemographic  characteristics of an individual patient may increase the perceived personal relevance of risk information, thereby increasing the likelihood that patients will process the tailored information with more deliberation, consideration, and evaluation [Typically, risk information about possible treatment side effects is generic and mostly based on the “average patient”, such as information presented in randomized controlled trials or patient reported outcome reports . Such avaluation , 18. In aluation  as well aluation , 21. Themessage format they should communicate tailored risk statistics, using for instance verbal and/or numerical formats [An important consideration for clinicians, health educators and patient tool developers is through which  formats . Verbal  formats . The pro formats –26. Anot formats , 26. Alt formats –27, it i formats  or incor formats –31.TableIn the present study, we will examine the impact of tailoring (tailored vs. generic risks) and message format  of risks of cancer treatment side effects on cancer patients’ risk interpretations. We will use estimation of probability, accuracy of estimation of probability, and perceived likelihood of occurrence as primary outcome variables. First, regarding the influence of tailoring, we expect that risks that are tailored will be perceived as more likely to occur than generic risks , 32.Compared to generic risks of treatment side effects, tailored risks will be perceived as more likely to occur.Second, given the growing importance of replication research in the empirical sciences for improving the reproducibility of earlier study’s results , we atte Compared to risks of treatment side effects presented in a verbal and numerical combined format, risks presented in a verbal-only format will be estimated as (a) higher) and (b) less accurate, and (c) perceived as more likely to occur.Third, regarding the combined effect of tailoring and message format, we assume that tailored risks expressed as words and numbers combined should improve peoples’ estimated risk accuracy even more compared to generic risk information –21. This Compared to generic risks of treatment side effects, tailored risks will be estimated as more accurate than generic risks, but only when the risks are presented in a verbal and numerical combined format.Finally, we will assess perceived personal relevance and perceived uncertainty of the risk information as secondary outcome measures, for which we propose the following two hypotheses: Tailored risks of treatment side effects will be perceived as more personally relevant than generic risks, regardless of the message format. Tailored risks of treatment side effects will be perceived as less uncertain than generic risks, regardless of the message format.25. We used estimation of probability, accuracy of estimation of probability, and perceived likelihood of occurrence as primary outcome variables, and perceived personal relevance and perceived uncertainty as secondary outcome variables.We used a 2 (tailoring: tailored vs. generic) × 2  × 2 (probability rate: low vs. high) mixed design, with repeated measures on the first and third factor. Participants were randomly assigned to one of the two message format conditions. We included probability rate as a methodological variable to investigate whether the effects of tailoring and message format are similar for high and low probability rates https://osf.io/ygchx), power calculations were conducted prior to data collection to determine our sample size using the program G*Power 3.1 [f = 0.10) with a 2 × 2 × 2 mixed design, a sample of 136 participants was needed . We therefore aimed for a minimum of 136 participants.Native Dutch adults between the ages of 18 and 70 who had been diagnosed with cancer in the past were selected from the scientific panel of the online cancer community platform Kanker.nl to participate in our study. We selected people who had been in a similar health situation before, since they are better able to imagine the given scenarios , thus enhancing the generalizability of our results . Patientower 3.1 . Previouower 3.1 , and medower 3.1 . To detereference class  to which the risk statistic applies. More specifically, tailored risks contained a reference class based on participants’ reported gender , age group (in 5-year bins between 15 and 69 years), and tumor stage (advanced colon cancer as stated in the scenario). For example: ‘This side effect is common ” ” Table . Generic)” Table .Table 2Dvaak in Dutch) for representing a low probability rate and ‘very common’ (zeer vaak in Dutch) for representing a high probability rate. Following the recommendations proposed by the European Commission, we used the corresponding natural frequency estimates ‘10 out of 100’ for representing a low probability rate and ‘40 out of 100’ for representing a high probability rate [Half of the participants received the risk only in words , and the other half in a combination of words and numbers . Within the verbal-only condition, we selected the verbal descriptors ‘common’ . This allowed us to include gender as a tailoring factor of the risk information. Participants were told that they were receiving a decision aid from their doctor including information about four possible side effects after adjuvant chemotherapy. Each description consisted of three elements: the name of the side effect, a short description of the side effect, and risk information about the likelihood of experiencing the side effect. This was followed by the assessment of the primary and secondary outcome measures. In the final part of the experiment, we measured participants’ subjective numeracy skills and prior history with chemotherapy and/or one of four mentioned the side effects. Participants were then debriefed about the main purpose of the experiment and thanked for their participation.estimation of probability was assessed using the question “What do you think is the probability you will experience this side effect”, measured as a percentage between 0 and 100 [accuracy of the estimation of probability was determined by computing the absolute difference between the actual risk of each side effect occurring and each participant’s estimated risk of that side effect occurring. Scores closer to zero were therefore more accurate . Th likely’ , 24.perceived personal relevance was assessed using the items “The risk information about the side effect was made personally for me” and “The way how the risk information was being presented was relevant to me”  [perceived uncertainty was assessed by asking the question “How uncertain do you think is this likelihood of experiencing this side effect after chemotherapy?”, measured on a 6-point scale, with 1 as ‘not at all’ and 6 as ‘extremely’ [We also included two secondary outcome variables. First,  agree’) . Second,tremely’ .Individual differences in subjective numeracy were assessed by the Subjective Numeracy Scale (SNS ), which p < .05. The study design, hypotheses, and analysis plan were pre-registered prior to data collection and analysis within the Open Science Framework (https://osf.io/j74dt/). Ethical approval was granted by the Research Ethics and Data Management Committee of the Tilburg School of Humanities and Digital Sciences of Tilburg University (ID REDC.2019.26).We conducted a 2 (within-subjects: tailoring) × 2 (between-subjects: message format) × 2 (within-subjects: probability rate) mixed-model multivariate analysis of variance (MANOVA).SD = 7.4), and the participants in both message format conditions were comparable in terms of sociodemographic and disease-related characteristics  clicked the link to launch the survey. Of those, 171 (91%) continued beyond the informed consent page, and 141 (75%) fully completed the survey  and perceived likelihood of occurrence , which, in turn, strongly correlated with participants’ accuracy of estimated probabilities .In both message format conditions, participants’ estimated probabilities strongly correlated with the accuracy of their estimated probabilities  = 6.25, p = .023, ηp2 = .04, and less accurate, F = 6.25, p = .014, ηp2 = .05, than generic risks  = 1.79, p = .183, ηp2 = .01. It should be noted that these tailoring effects were not found when controlling for individual differences in numeracy and prior history with the side effects .There was a significant main effect of tailoring on the estimation of probabilities and accuracy of estimation of probabilities. Tailored risks were estimated as higher, F1, 12 = 6.25, F = 69.82, p < .001, ηp2 = .36, accuracy of estimation of probabilities, F = 64.26, p < .001, ηp2 = .34, and perceived likelihood of occurrence, F = 30.27, p < .001, ηp2 = .20. The results therefore suggest that risks presented in a verbal-only format were estimated as higher (H2a), less accurate (H2b), and perceived as more likely to occur (H2c) than risks presented in a verbal and numerical combined format. These message format effects were also found when controlling for individual differences .As hypothesized, there was a significant main effect of message format on the estimation of probabilities, F, 125 = 6F = 7.82, p = .006, ηp2 = .06. Simple effect analysis showed that tailored risks were estimated as less accurate than generic risks in the verbal-only condition, (p < .001), but not in the combined condition (p = .833). This is in contrast to our hypothesis (H3), for which we expected tailored risks to be estimated as more accurate compared to generic risks, but only when expressed as words and numbers combined. There was also a significant interaction effect on the estimation of probabilities, F = 7.21, p = .008, ηp2 = .06. Simple effect analysis revealed that tailored risks were estimated as higher than generic risks in the verbal-only condition (p = .001), but not in the combined condition (p = .789). Overall, these significant interaction effects were found for both probability rates, and when controlling for individual differences  = 1.79, p = .183, ηp2 = .01. Figure There was a significant interaction effect between tailoring and message format on the accuracy of estimation of probabilities, F, 125 = 7F = 19.11, p < .001, ηp2 = .13  = 2.36, p = .127, ηp2 = .02, and probability rate, F < 1. Regarding perceived uncertainty, there was a significant interaction effect between tailoring and message format, F = 6.23, p = .014, ηp2 = .05. Simple effects analysis showed that tailored risks in the verbal-only condition were perceived as less uncertain than generic risks (p = .007), but not in the verbal and numerical combined condition (p = .436), which partly confirms H5. Finally, risks with low probability rates were perceived as more uncertain than risks with high probability rates, F = 11.01, p < .001, ηp2 = .09.As hypothesized H4), participants perceived tailored risks as more personally relevant than generic risk information about side effects, , partici3 Tables . This efThe current study demonstrates that message format matters when communicating tailored risk information of treatment side effects. We found that communicating tailored side effect risks leads to higher and less accurate risk estimates compared to generic risks, but only when the risks were communicated using words-only. Such differences were not found in the combined verbal and numerical condition. This suggests that communicating about side effect risks in words-only allows patients to overestimate and even inaccurately estimate their tailored risks , 41, 42.However, tailored risks in a verbal and numerical combined format did not lead to more accurate risk estimates compared generic numerical risk information –21. A poFinally, in both message formats, tailored risks are perceived as more personally relevant than generic risks, which is in line with past studies on tailoring effects in health communication . In addiA first limitation is that the research design uses a hypothetical decision-making scenario instead of a real decision-making scenario. To partially compensate for this, our sample consisted of cancer patients and survivors who were recruited from a Dutch cancer patient panel. Often, scenario-based experimental studies on effective risk communication strategies are conducted in student samples , who maAnother limitation is that we tailored the risks based on a limited number of patient characteristics in a non-interactive way, to keep the experiment manageable and the results generalizable. Clinical prediction models in oncology settings typically utilize a larger variety of patient and tumor characteristics in decision-making  that is more extensive than we have dealt with in our study. Using such an interactive prediction modelling tool in which participants can enter their own personal and disease-related characteristics and see the impact of each characteristic on their personal risk could influence patients’ risk perception . DespiteFinally, we only compared risks communicated through words or a combination of words and numbers, and did not consider the potential added value of visual aids as another message format. A plethora of research suggests that visual aids may increase understanding and perception of risk information , 49, 50.Despite these limitations, our findings have implications for research and practice. First, in line with guidelines and best practices for communicating complex medical data and risks in daily clinical practice and patient decision aids , 42, 51,When communicating tailored risk information of treatment side effect to patients, using a combination of words and numbers will lead to more accurate risk estimates than when using words only. Although we found no evidence that tailoring of numerical risks leads to even more accurate risk estimates, doing so with verbal labels alone may have a negative impact on patients’ (accuracy) of estimation of risks. Given the strong movements toward personalized medicine and patient-centered healthcare, future research will have to determine whether other ways of presenting tailored risk information, such as comparative risk information or visual aids promote effective communication of tailored risks during cancer treatment decision-making.Additional file 1: MANOVA and MANCOVA outcomes.