Prevalence of Ketosis Detected by an Electronic Cow Side Test and its Impact to Days Open on Dairy Cows

The study aimed to resolve and confirm the previous results of subclinical and clinical ketosis prevalence in northwestern Croatia detected by an electronic cow side test and its impact on days open in dairy herds. Cows (N=559) 2-8 years old from 96 farms located in northwestern Croatia were included in the study. The average milk yield was 7327.80 ± 968.21 kg. The cows were classified into two separate groups: ketotic cows group KET (n=73) with BHBA blood concentration of ≥1.4 mmol/L, and negative NEG (n=486) group with serum BHBA level of <1.4 mmol/L. One droplet of blood from the caudal vein was collect from randomly selected cows in the period 7 to 15 days after parturition. BHBA level determined with Precision Xceed BHBA devices. The period from parturition to first insemination (days open to the first (artificial) insemination, DOFI) was shorter in NEG than in the POS group (110.56±10.65 days vs.114.82±12.23 days, respectively) such as period from parturition to successful conception (days open to successful conception, DOSC), (139.97±15.18 days vs. 127.99±15.87 days, respectively). The prevalence of clinical and subclinical ketosis was 15.02%. Ketosis significantly prolonged days open to first artificial insemination and days open to successful conception in cows from northwestern Croatia.

Determination of oroxin A, oroxin B, oroxylin A, oroxyloside, chrysin, chrysin 7-O-beta-gentiobioside, and guaijaverin in mouse blood by UPLC-MS/MS and its application to pharmacokinetics

In this study, a UPLC-MS/MS method was developed to measure the concentrations of the flavonoids oroxin A, oroxin B, oroxylin A, oroxyloside, chrysin, chrysin 7-O-beta-gentiobioside, and guaijaverin in the blank mouse blood, and the method was then used in the measurement of the pharmacokinetics of the compounds in mice. Oroxin A, oroxin B, oroxylin A, oroxyloside, chrysin, chrysin 7-O-beta-gentiobioside, and guaijaverin were administered intravenously at a dose of 5 mg kg−1, and the mouse blood (20 μL) was withdrawn from the caudal vein 0.08333, 0.25, 0.5, 1, 2, 4, 6, 8, and 10 h after administration. The mobile phase used for chromatographic separation by gradient elution was composed of acetonitrile and water (0.1% formic acid). The analytes were detected by operating in electrospray ionization (ESI) positive-ion mode using multiple reactions monitoring (MRM). The intra-day and inter-day accuracy ranged from 86.2 to 109.3%, the intra-day precision was less than 14%, and the inter-day precision was less than 15%. The matrix effect ranged from 85.3 to 111.3%, and the recovery of the analytes after protein precipitation were all above 78.2%. This method had the advantages of high sensitivity, accuracy, and recovery, and it had excellent selectivity, which enabled it to be applied to measuring the pharmacokinetics of the analytes in mice.

Fish disease study of Asian seabass (Lates calcarifer) in a floating marine cage: Endoparasite and blood profile

Hidayati D, Nurindra AR, Abdulgani N, Setiawan E, Maulidina N, Syahroni N, Mulyadi Y. 2021. Fish disease study of Asian seabass (Lates calcarifer) in a floating marine cage: Endoparasite and blood profile. Biodiversitas 22: 4505-4511. The Indonesian government has multiple targets and priority programs to increase aquaculture productivity in 2021. Hence, the development of mariculture commodities such as Asian seabass (Lates calcarifer) in one of the potential coastal areas, Sendang Biru, is strategic. However, sustainable productivity needs good management, including the health monitoring of fish. This study aimed to determine the condition of seabass under mariculture in a floating cage in Sendang Biru, Malang, Indonesia. Thirty seabass (Lates calcarifer) from 0 to 60 days after stocking age (ASE) were taken from a Sendang Biru floating cage. Blood and intestine samples were taken five times from day 0 to day 60 ASE. Blood samples were carried out through the caudal vein and observed using a hemocytometer. Endoparasite analysis was performed by fish intestine examination from the anterior intestine to the posterior rectum. Pseudoterranova and Diphyllobothrium were found with 10% and 3.33% prevalence, respectively. The intensity of Pseudoterranova was one individual/fish, and that of Diphyllobothrium was 2.33 individuals/fish. The highest mean of total erythrocyte count (TEC) found in 0 day ASE was at the level of 0.82±0.55×106 cells/mm3, and the lowest was found in 60 days ASE (0.51±0.24×106 cells/mm3). The highest mean of total leucocyte count (TLC) was 9.20±3.69x104 cells/mm3, at day 0, and leukocyte levels tended to decrease until day 60 ASE (7.74±3.43×104 cells/mm3). Overall, despite the TEC and TLC tending to decrease from 0 to 60 days ASE, in statistically indicated no significant differences in TLC at different ASEs (p > 0.05). The hematological profile indicated the health statuses of the cultured Asian seabass in the current study were in a condition of anemic and infected.

Characterizing the cardiovascular phenotype of a new zebrafish model of Marfan syndrome

Background Marfan syndrome (MFS) is a rare disease caused by a defect in the fibrillin-1 gene (FBN1), with potentially severe cardiovascular manifestations. MFS patients are particularly susceptible to a progressive aortic dilation leading to potential dissection and wall rupture. No causal treatment for the disease is available and current medical treatment is aimed at slowing aortic disease progression to minimize severe complications. When indicated, surgical repair of the aortic defect is performed. Although these strategies have clearly led to improved survival, some patients still present with fatal complications. Purpose To generate a new flexible zebrafish model of MFS to gain a better understanding of the underlying pathophysiological mechanisms and to find new treatment options. Methods We used the CRISPR/Cas9 method to disrupt the 3 fibrillin genes in zebrafish (fbn1, fbn2a, and fbn2b). The Tg(kdrl:EGFP) reporter was used to visualize cardiovascular structure by fluorescent microscopy up to 8 days post fertilization (dpf). A subset of embryos was treated with the myosin inhibitor 2,3-butanedione monoxime (BDM). Results We found that zebrafish lacking fbn1 and/or fbn2a do not show any detectable phenotype during development. No evidence of induction of genetic compensation was found in these mutant lines. Zebrafish deficient in fbn2b however do show strong phenotypes, including fully penetrant finfold atrophy (Fig. 1A-B and E, arrowhead). On average 50% of homozygous fbn2b mutant (fbn2b−/−) zebrafish embryos show endocardial detachment (Fig. 1C-D; a:atrium, v:ventricle), leading to vascular embolism, pericardial edema (Fig. 1B, arrow), loss of blood flow, and ultimately death at 7–9 dpf. Interestingly, fbn2b−/− without endocardial detachment survive normally, but develop a dilated bulbus arteriosus phenotype during larval stages (Fig. 1F-G, arrow; 1H: diameter during minimal and maximal distension, *: P&lt;0.05 and ***: P&lt;0.001 by Sidak's post-test after Two-Way ANOVA). This anatomical structure is strongly related to the aortic root in humans, which is the predominant location of aortic dilation in MFS. All fbn2b−/− embryos show abnormal early development of the caudal vein as a cavernous structure lacking vessel integrity (Fig. 2, arrowheads). This phenotype resolves in embryos retaining normal blood flow. We found that fbn2b−/− embryos raised in BDM to inhibit blood flow show a more severe caudal vein phenotype than wild-type (WT) controls (Fig. 2, yellow line: severe vascular dilation). Conclusion Loss of fbn2b, but not the other fibrillin genes, in zebrafish results in cardiovascular manifestations overlapping with MFS. These data indicate that fbn2b−/− zebrafish can be a relevant model to explore the mechanisms leading from fibrillin deficiency to the cardiovascular symptoms observed in MFS. Our preliminary results suggest that there is an interplay between fibrillin deficiency and biomechanical signaling. FUNDunding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Fund Baillet Latour Grant for Medical Research Figure 1. Phenotype of fbn2b−/− larvae Figure 2. Effect of blood flow in fbn2b−/−

Protective Effect of Exosomes Derived from Bone Marrow Mesenchymal Stem Cells Against AD-Like Behaviors in Mice: Involvement with Regulating Glial Activation and its Associated Neuroinflammation and BDNF-Related Neuropathological Changes in the Hippocampus

Background: Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive decline in cognitive ability. Exosomes derived from bone marrow mesenchymal stem cells (BMSC-exos) are extracellular vesicles that can execute the function of bone marrow mesenchymal stem cells (BMSCs). Given the versatile therapeutic potential of BMSC and BMSC-exos, especially the neuroprotective effect, the aim of this study is to investigate the potential effect of BMSC-exos on the AD-like behavioral dysfunction in mice and explore the possible molecular mechanism.Methods: BMSCs were isolated from the femur and tibia of adult C57BL/6 mice, purified and sorted via flow cytometry technology, and cultured in vitro. The BMSC-exos were extracted and identified via transmission electron microscopy, and Western Blot was used to detect exosomes labeled proteins. A sporadic AD mouse model was established by intracerebroventricular injection of streptozotocin (STZ). 6 weeks later, BMSC-exos were given via lateral ventricle injection or caudal vein injection lasting for five consecutive days, and the control mice were intracerebroventricular given equal volume of solvent. The behavioral performance were observed via the Open field test (OFT), elevated plus maze test (EPM), novel object recognition test (NOR), Y maze test (Y-maze), and tail suspension test (TST). The mRNA and protein expressions of IL-1β, IL-6, and TNF-α in the hippocampus were measured via quantitative polymerase chain reaction (QPCR) and Western Blot, respectively. Meanwhile, the protein expression of Aβ1-42, BACE, p-Tau (Ser396), Synaptotagmin-1, Synapsin-1, and brain-derived neurotrophic factor (BDNF) in the hippocampus was detected using Western blot, and the expression of GFAP, IBA1, and DCX in the hippocampus was measured via immunofluorescence staining.Results: Lateral ventricle administration, but not caudal vein injection of BMSC-exos could improve the AD-like behaviors in STZ-injected mice model, as indicated by the increased number of rearing, increased frequency to the central area, and increased duration and moving distance in the central area in the OFT, together with improved preference index of the novel object in the NOR. Moreover, the hyper-activation of microglia and astrocytes in the hippocampus of the model mice were inhibited after treatment with BMSC-exos via lateral ventricle administration, accompanied by the reduced expression of IL-1β, IL-6, TNF-α, Aβ1-42, and p-Tau, and upregulated protein expression of synapse-related proteins and BDNF. Furthermore, results of Pearson test showed that the preference index of the novel object in the NOR was positively correlated to the hippocampal expression of BDNF, but negatively correlated with the expression of GFAP, IBA1, and IL-1β. Apart from a positive correlation between the hippocampal expression of BDNF and Syt-1, BDNF abundance was found negatively correlated with the markers of glial activation and the expression of inflammatory cytokines, Aβ, and p-tau, which were characteristic neuropathological features of AD.Conclusions: Lateral ventricle administration, but not caudal vein injection of BMSC-exos can improve the AD-like behavioral performance in STZ-injected mice, the mechanism of which might be involved with regulating glial activation and its associated neuroinflammation and BDNF-related neuropathological changes in the hippocampus.

Does the Site of Blood Collection in Fish Affect Haematological and Blood Biochemical Results?

The values of haematological and selected blood plasma biochemical parameters of juvenile common carp (Cyprinus carpio Linnaeus, 1758) were compared between blood samples taken from caudal vein and heart to evaluate the influence of blood sampling body site on the obtained results in two groups of fish of different blood sampling order: I – first by caudal and then by cardiac puncture, II – first by cardiac and then by caudal puncture. The obtained results revealed statistically significant (p<0.05) differences only in group I where red blood cell (RBC) count was higher in caudal vein blood, while haematocrit (Ht) value, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), total protein (TP) concentration, and magnesium (Mg) level were higher in cardiac blood samples. No statistically significant differences occurred in white blood cell (WBC) count, differential leukocyte count or erythrocyte morphology based on stained blood smears. The obtained results showed that blood sampling body site may affect the results of haematological and plasma biochemical analyses.

Establishment of a male Wistar rat model of nanobacteriainduced kidney stones

Purpose: To establish a male Wistar rat model of nanobacteria (NB)-induced kidney stones. Methods: Sixty male Wistar rats were randomly divided into control group (NC group) given caudal vein injection of saline + saline gavage, and NB-induced stone group (NBS group) given caudal vein injection of NB + saline gavage. Results: Compared with NC, serum creatinine, blood uric acid, urea nitrogen and urinary calcium levels in NBS group increased between weeks 3 and 8 (p < 0.05). Kidney index (kidney weight/body weight ratio) in the NBS group was higher than that in NC group from weeks 8-10. At week 8, urine pH and serum phosphorus in NBS group were higher than those in NC group (p < 0.05). Between weeks 6 and 7, serum calcium in NBS group was higher than that in NC group (p < 0.05). Calcium crystals in NBS rats were distributed mostly in the distal and proximal convoluted tubules. However, no such crystals were observed in NC rats. Similarly, no such pathological changes were seen in the renal tissue of NC group. Calculus analysis showed that stone formation was higher in NBS group than in NC group (p < 0.05). There was no significant difference in micro-CT between the two groups (p ˃ 0.05). Conclusion: The successful establishment of the Wistar rat kidney stone model using NB cultured from urine of upper urinary tract stone patient is potentially useful for further etiological studies on kidney stone formation.

Effect of Diets With Different Lysine Levels on the Hemograms of Juvenile Tambaqui Colosssoma Macropomum (CUVIER, 1818)

Blood transports lysine and other nutrients derived from the diet and ensures good health and greater productivity for the fish. Therefore, this study aimed to verify the hematological behavior of juvenile tambaqui Colossoma macropomum nourished with levels of 9.72, 12.84, 15.96, 19.08 and 22.20 g.Kg-1 of total lysine, corresponding, respectively, to the supplementation levels with L-lysine: 0.00, 4.00, 8.00, 12.00, 16.00 and 20.00 g.Kg-1 of L-lysine. The blood of the fish was collected in the initial and final periods by puncture of the caudal vein. The variables were validated by ANOVA and Tukey's test (p< 0.05). No differences were found for hemoglobin, hematocrit, CHCM, leukocytes of the eosinophil and monocyte types, and thrombocytes (p> 0.05). The changes observed in total leukocytes were not attributed to diets (p= 0.00). Means of CMV, HCM, lymphocytes and neutrophils, varied over the initial period, but not in regard to different lysine levels (p< 0.05). Thus, it is concluded that the varying lysine levels of the diets did not compromise the hematological parameters analyzed.

Cytogenetic evaluation of α, β-amirina obtained in resin of Protium heptaphylum (Aubl.) Marchand in polychromatics erythrocytes of mice swiss.

The Amazon Rainforest has a great variety of medicinal plants, among them we can highlight the “Almecegueira” or “Breu Branco” (Protium heptaphylum) in Portuguese, the producer of a greenish-white resin that hardens when it touches the air, known by its gastroprotective and anti-inflammatory effects. These effects are attributed to a triterpene mixture of α and β amirine, predominant in the resin. The purpose of the study is to obtain a cytogenetic profile to the α, β-amirine mixture obtained in the resin of P. heptaphylum. For this, the micronucleus test was used in peripheral blood and bone marrow; administering solution in Swiss mice with the dosages of 1mg/Kg, 3mg/Kg, and 10mg/Kg, diluted in 5% DMSO, the effects were observed in 24h and 48h after the treatment. For the test in peripheral blood the mice’s caudal vein was punctured, while for the bone marrow test, the femurs of the animals were obtained from which bone marrow samples were taken. It was found that in peripheral blood, the administration of the compounds did not cause genotoxicity in 24h and 48h, in contrast, antigenotoxicity was, for concentrations 1; 3 and 10mg/kg, respectively 10%; 12%; 67% in 24h and 9%; 15%; 73% in 48h. In the bone marrow, no genotoxicity was observed, as for antigenotoxicity was observed that for concentrations 1; 3 e 10mg/kg the percentage of reduction was respectively: 11%, 15%, and 30% in 24h and 13% 16% 33% in 48h. It is concluded that the studied compound can be an alternative for treatments in the future since it presents low toxicity and high antigenotoxic potential.