A Pilot Study of the Role of Selected Biomarkers of Kidney Injury in Dogs with Dilated Cardiomyopathy.

Heart and kidney diseases are among the most frequent medical conditions diagnosed in small animals. Due to the functional interconnection between these organs, the concept of the cardio-renal axis has been developed. In this context, renal disease or dysfunction often occurs secondary to heart diseases, such as dilated cardiomyopathy (DCM). DCM is the most common cardiomyopathy and a leading cause of mortality in large-breed dogs. Traditional biomarkers like creatinine or symmetric dimethylarginine concentration are not always effective, especially in the early stages of the disease, underscoring the need for more sensitive markers of renal impairment during heart failure (HF). This study aimed to evaluate the efficacy of selected biomarkers as indicators for early kidney damage in dogs with stage B2 DCM. We measured serum concentrations of cystatin C, KIM-1 (kidney injury molecule-1), and NGAL (neutrophil gelatinase-associated lipocalin) and their ratios to creatinine, analyzing their diagnostic values. Cystatin C was quantified using a sandwich enzyme immunoassay, while KIM-1 and NGAL were measured with enzyme-linked immunosorbent assay kits designed for canine diagnostics. The concentrations were indexed against serum creatinine. The study included 26 dogs: 9 with HF and 17 healthy controls. The mean ± standard deviation for healthy dogs for cystatin C, cystatin C/creatinine ratio, KIM-1, KIM-1/creatinine ratio, NGAL, and NGAL/creatinine ratio were 0.24 ± 0.04, 0.26 ± 0.07, 0.61 ± 0.07, 0.67 ± 0.13, 2.76 ± 1.8, and 2.79 ± 1.81, respectively. For DCM dogs, these values were 0.27 ± 0.1, 0.32 ± 0.12, 0.61 ± 0.08, 0.69 ± 0.17, 6.46 ± 5.22 (p = 0.02), and 7.99 ± 6.53 (p = 0.04). This study's findings suggest that during the asymptomatic phase of DCM, only NGAL concentration and the NGAL/creatinine ratio may serve as diagnostic markers for early-stage kidney injury.

The Effect of Orally Administered Multi-Strain Probiotic Formulation (Lactobacillus, Bifidobacterium) on the Phagocytic Activity and Oxidative Metabolism of Peripheral Blood Granulocytes and Monocytes in Lambs.

Probiotic feed additives have attracted considerable research interest in recent years because the effectiveness of probiotics can differ across microbial strains and the supplemented macroorganisms. The present study was conducted on 16 lambs divided equally into two groups (C-control and E-experimental). The examined lambs were aged 11 days at the beginning of the experiment and 40 days at the end of the experiment. The diet of group E lambs was supplemented with a multi-strain probiotic formulation (Lactobacillus plantarum AMT14, Lactobacillus plantarum AMT4, Lactobacillus rhamnosus AMT15, and Bifidobacterium animalis AMT30), whereas group C lambs did not receive the probiotic additive. At the beginning of the experiment (day 0) and on experimental days 15 and 30, blood was sampled from the jugular vein to determine and compare: phagocytic activity (Phagotest) and oxidative metabolism (Phagoburst) of peripheral blood granulocytes and monocytes by flow cytometry. An analysis of the phagocytic activity of granulocytes and monocytes revealed significantly higher levels of phagocytic activity (expressed as the percentage of phagocytic cells and mean fluorescence intensity) in lambs that were administered the multi-strain probiotic formulation compared with lambs in the control group. The probiotic feed additive also exerted a positive effect on the oxidative metabolism of both granulocytes and monocytes (expressed as the percentage of oxidative metabolism and mean fluorescence intensity) after stimulation with Escherichia coli bacteria and with PMA (4-phorbol-12-ß-myristate-13-acetate). These findings suggest that the tested probiotic formulation may have a positive effect on the immune status of lambs.

The Role of Selenitetriglycerides in Enhancing Antioxidant Defense Mechanisms in Peripartum Holstein-Friesian Cows.

The transition period in high-yielding dairy cows is a critical phase marked by an elevated risk of oxidative stress. This study evaluated the effect of oral selenitetriglyceride supplementation on oxidative stress management in periparturient cows. A controlled experiment was conducted on 12 cows, divided into two groups: the experimental group (STG) received selenitetriglycerides (0.5 mg Se/kg BW), while the control group (CON) was given a placebo, starting 12 days before calving until the calving day. Blood and liver tissue samples were collected at predetermined intervals around the time of parturition. The study observed a significant increase in serum selenium levels and NEFA stabilization in the STG group compared with the control. Antioxidant parameters indicated elevated GSH-Px and CAT concentrations in the STG group. Liver gene expression analysis revealed a significant increase in SOD2 mRNA levels in the STG group (FC = 4.68, p < 0.01). Conversely, GSH-Px3 expression significantly decreased (FC = 0.10, p < 0.05) on the 7th day postpartum in the CON group. However, SOD1, SOD3, and CAT expressions remained stable in both groups. These findings highlight the beneficial role of selenitetriglycerides in enhancing antioxidant capacity and influencing specific gene expressions associated with oxidative stress management in dairy cows during the peripartum period.

Progesterone-independent endometrial mRNA expression in dairy cows with clinical or subclinical endometritis.

Up to 50 % of dairy cows fail to resolve uterine involution and develop chronic clinical (CE) or subclinical endometritis (SE) 21 days after calving. Clinical endometritis is associated with purulent discharge, while SE is not associated with overt clinical signs. Along with numerous knowledge gaps related to its pathogenesis, SE does not allow for a straightforward and effective therapy. Therefore, it is crucial to unravel differences in the expression of genes among healthy, CE, and SE cows. This might contribute to the discovery of new drug candidates and, in consequence, a potentially effective treatment. In the present study, cows between 21 and 28 days postpartum (PP) were examined using vaginoscopy for the presence of vaginal discharge and endometrial cytology for the determination of the endometrial polymorphonuclear cell (PMN) percentage. Next, an endometrial biopsy sample was taken to investigate the expression of 13 selected candidate genes by qPCR. Uterine health status was assigned to healthy (absence of abnormal vaginal discharge and ≤5 % PMN, n = 13), SE (absence of abnormal vaginal discharge and >5 % PMN, n = 30), and CE (mucopurulent or purulent vaginal discharge and >5 % PMN, n = 9). At the same time, a blood sample was collected to assess serum progesterone concentration and to categorize cows as low (≤1 ng/mL) or high (>1 ng/mL) in progesterone. High expression of IL1B, IL6, IL17A, CXCL8, PTGES, PTGS1, PTGS2, and INHBA genes and low expression of FST was noted in the endometrium of CE compared to healthy cows. Increased endometrial INHBA expression was observed in both SE and CE compared to healthy cows. Interestingly, greater expression of PTGES and PRXL2B genes and lower expression of PTGS2 were characteristic of SE versus CE or healthy. Among cows with no overt clinical symptoms of uterine disease (healthy and SE), the endometrial expression of IL1 B, CXCL8, and PTGES was greater in cows with high versus low serum progesterone. Several genes were differentially expressed among healthy, SE, and CE cows indicating different pathways for the development of different uterine diseases. In conclusion, we found progesterone-independent SE markers, which suggests that low endometrial PTGS2 expression may be indicative of an inadequate immune response and thus contribute to the pathogenesis of SE.

Prevalence of subclinical endometritis in dairy cows that recovered after treatment of clinical endometritis with cephapirin and PGF2α.

The aims of our study were to evaluate (1) the prevalence of subclinical endometritis (SE) in clinically cured cows after treatment of clinical endometritis (CE) with uterine cephapirin infusion or with systemic application of PGF2α and after self-healing, (2) the prevalence of SE in clinically cured cows in relation to the type of vaginal discharge. The study was conducted on 222 Polish Holstein Friesian cows with CE diagnosed by vaginoscopy and ultrasound. The animals were randomly allocated to one of three groups: Group 1 (n = 72): intrauterine cephapirin infusion; Group 2 (n = 73): single intramuscular injection of dinoprost; and Group 3 (n = 77): left untreated as a control. All cows were reexamined two weeks later (Exam 1). If there was still CE at this examination, these cows were treated using the same method as previously and were examined 2 weeks later (Exam 2). Cows still having CE at the second examination were examined two weeks later (Exam 3). Cows without signs of CE at Exams 1, 2 and 3 were considered cured, and endometrial samples from the uteri were collected by cytobrush to diagnose SE using cytological evaluation of PMN percentage. The threshold for SE was set at ≥ 5% PMNs. Two cows each from Groups 1 and 3 and three cows from Group 2 were not clinically recovered at Exam 3 and were excluded from the analysis. In total, SE was diagnosed in an average of 40.9% of clinically cured cows. The prevalence of SE in Group 1, 2 and 3 was 35.0%, 47.1% and 40.0%, respectively (p > 0.05). There were significantly higher (p < 0.05) SE cases after treatment of CE associated with purulent vaginal discharge than with mucopurulent discharge. In relation to the method of treatment, the prevalence of SE was significantly higher (p < 0.05) in cows treated with cephapirin with purulent vaginal discharge than with mucopurulent vaginal discharge. In conclusion, the study showed a high prevalence of SE in cows clinically cured after treatment of clinical endometritis with cephapirin or PGF2α and after self-healing. The prevalence of SE was significantly higher in cows with purulent vaginal discharge than with mucopurulent vaginal discharge. Therefore, in cows clinically recovered from clinical endometritis, the prevalence of SE should always be considered a negative consequence, and the resulting management of this uterine disease should be implemented at the herd level.

Identification of Serum-Predictive Biomarkers for Subclinical Mastitis in Dairy Cows and New Insights into the Pathobiology of the Disease.

Targeted direct injection/liquid chromatography coupled to tandem mass spectrometry-based metabolomics was employed to identify metabolite alterations that could differentiate subclinical mastitis (SCM) from control (CON) dairy cows at -8, -4, disease diagnosis, +4 and +8 wks relative to parturition. We identified and measured 128 metabolites in the serum. Univariate analysis revealed significant alterations of serum metabolites at all five time points studied. By applying multivariate analyses including principle component analysis and partial least squares-discriminant analysis, some of the metabolites were found to have the strongest power for discriminating the SCM from CON cows. The top five metabolites with the greatest variable importance in projection values were selected as potential biomarkers for SCM. A set of five serum metabolites including lysine, ornithine, isoleucine, LysoPC a C17:0, and leucine at -8 wks and five other metabolites including lysine, leucine, isoleucine, kynurenine, and sphingomyelin (SM) C26:0 at -4 wks prepartum were determined as predictive biomarkers for SCM, which provided highly predictive capabilities with AUC (area under the curve) at 1.00. Five metabolites including lysine, leucine, isoleucine, kynurenine, and SM C26:1 in the serum were identified as diagnostic biomarkers for SCM with the AUC of 1.00. Moreover, we observed that distinct metabolic pathways were affected in SCM cows including lysine degradation, biotin, cysteine, methionine, and glutathione metabolism, valine, leucine, and isoleucine biosynthesis and degradation, and aminoacyl-tRNA biosynthesis prior to and during the occurrence of the disease. Results of this study showed that metabolomics analyses can be used to identify susceptible cows to SCM starting from -8 and -4 wks prepartum and that blood can be used to diagnose cows with SCM.

A Targeted Serum Metabolomics GC-MS Approach Identifies Predictive Blood Biomarkers for Retained Placenta in Holstein Dairy Cows.

The retained placenta is a common pathology of dairy cows. It is associated with a significant drop in the dry matter intake, milk yield, and increased susceptibility of dairy cows to metritis, mastitis, and displaced abomasum. The objective of this study was to identify metabolic alterations that precede and are associated with the disease occurrence. Blood samples were collected from 100 dairy cows at -8 and -4 weeks prior to parturition and on the day of retained placenta, and only 16 healthy cows and 6 cows affected by retained placenta were selected to measure serum polar metabolites by a targeted gas chromatography-mass spectroscopy (GC-MS) metabolomics approach. A total of 27 metabolites were identified and quantified in the serum. There were 10, 18, and 17 metabolites identified as being significantly altered during the three time periods studied. However, only nine metabolites were identified as being shared among the three time periods including five amino acids (Asp, Glu, Ser, Thr, and Tyr), one sugar (myo-inositol), phosphoric acid, and urea. The identified metabolites can be used as predictive biomarkers for the risk of retained placenta in dairy cows and might help explain the metabolic processes that occur prior to the incidence of the disease and throw light on the pathomechanisms of the disease.

Effect of a single, oral administration of selenitetriglycerides, at two dose rates, on blood selenium status and haematological and biochemical parameters in Holstein-Friesian calves.

BACKGROUND: Selenitetriglycerides are biologically active, organic forms of selenium formed as a result of the modification of selenic acid and sunflower oil. Studies in rats have shown that they are well absorbed and of low toxicity. There are no published studies on selenitetriglycerides supplementation in calves. RESULTS: In this study, selenitetriglycerides were administered once orally on the 2nd day of life at a dose of 0.5 or 1 mg Se/kg body weight to each of six Holstein-Friesian calves while six control calves were not supplemented. Blood for determination of selenium concentration, glutathione peroxidase activity, haematological parameters, aspartate aminotransferase, creatine kinase, and lactate dehydrogenase activities and glucose, total protein, albumin, triglycerides, cholesterol, urea, and creatinine concentration was collected before supplementation (day 0) and 1, 2, 5, 10 and 14 days after supplementation. Selenitetriglycerides administration increased (P < 0.01) serum selenium concentration in supplemented calves as early as day1, from a mean of 63.4 to 184.22 µg/l in calves receiving selenium at a dose of 0.5 mg/kg BW, and from 63.17 to 200.33 µg/l in calves receiving 1 mg/kg. Serum selenium concentrations remained significantly higher compared to the control group throughout the experiment. Glutathione peroxidase activity was higher in supplemented than control calves, significantly so in animals receiving the 1 mg/kg dose of Se on the 10th and 14th days (P < 0.05). There were no significant differences in the haematological and biochemical parameters between the groups. CONCLUSIONS: This experiment showed that supplementation with selenitetriglycerides could significantly improve blood selenium status in calves without adverse effects on haematological or biochemical parameters. These findings are essential prerequisites for future studies on selenitetriglycerides supplementation to manage clinical selenium deficiency in calves.

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

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

Is similarity in Major Histocompatibility Complex (MHC) associated with the incidence of retained fetal membranes in draft mares? A cross-sectional study.

The failure of the maternal immune system to recognize fetal antigens and vice versa due to MHC similarity between the foal and its dam might result in the lack of placental separation during parturition in mares. The aim of the study was to investigate the influence of MHC similarity between a mare and a foal on the incidence of retained fetal membranes (RFM) in post-partum mares. DNA was sampled from 43 draft mares and their foals. Mares which failed to expel fetal membranes within three hours after foal expulsion were considered the RFM group (n = 14) and mares that expelled fetal membranes during the above period were the control group (n = 29). Nine MHC microsatellites of MHC I and MHC II were amplified for all mares and foals. MHC compatibility and MHC genetic similarity between mares and their foals was determined based on MHC microsatellites. The inbreeding coefficient was also calculated for all horses. The incidence of RFM in the studied population was 33%. Compatibility in MHC I and MHC II did not increase the risk of RFM in the studied population of draft mares (P>0.05). Differences in MHC similarity at the genetic level were not observed between mare-foal pairs in RFM and control group (P>0.05). We suspect that RFM in draft mares may not be associated with MHC similarity between a foal and its dam. Despite the above, draft horses could be genetically predisposed to the disease.

Applicability of the Protein-lipid Profile and Activity of Lactate Dehydrogenase Isoenzymes for Diagnosing Nutritional Muscular Dystrophy in Calves.

INTRODUCTION: In calves, hyposelenosis degenerates skeletal muscles in different parts of the body. The extent of damage to muscle cells can be diagnosed by determining the activity of creatine kinase (CK), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH). The aim of this study was to analyse variations in the serum levels of LDH isoenzymes in calves with nutritional muscular dystrophy (NMD), to determine the applicability of this parameter for diagnosing NMD, and to describe the influence of hyposelenosis on total protein (TP), triglyceride (TG), and cholesterol (CHOL) levels. MATERIAL AND METHODS: Two groups of calves (n = six animals per group) were used. After birth, control group calves (SC) were intramuscularly administered 10 ml of a preparation containing selenium (Se) and vitamin E, and experimental group animals (SE) that were not injected. Blood was collected after 5, 15, and 25 days, and the concentrations of Se, vitamin E, TP, TG, and CHOL and the activity of glutathione peroxidase (GSH-Px), CK, and LDH fractions were determined. RESULTS: Hypocholesterolaemia and elevated TG levels were found in SE group calves whose LDH fractions revealed a significant increase in LDH4 and LDH5 activity and a decrease in LDH1 activity when electrophoretically separated. CONCLUSION: Nutritional muscular dystrophy is accompanied by hypocholesterolaemia and elevated TG levels caused by muscle lipolysis. LDH4 and LDH5 activity parameters assist early diagnosis of NMD in calves.