The use of COLD‐PCR,DHPLC and GeneScanning for the highly sensitive detection of c‐KIT somatic mutations in canine mast cell tumours

The conventional polymerase chain reaction (PCR)/sequencing methods may be poorly suited for the detection of somatic mutations in canine mast cell tumour (MCT) samples owing to limited sensitivity. This study was aimed at establishing novel and more sensitive methods, assessing their limit of detection and comparing their sensitivity with conventional methods.Two different ‘driver’ somatic mutations of c‐KIT, together with the wild‐type counterparts, were cloned in plasmids to prepare standard samples with known concentrations of mutated alleles in a background of wild‐type alleles; the plasmids standards were assayed using either conventional or novel, highly sensitive technique. Conventional PCR/sequencing showed a sensitivity of 50–20%. Conversely, all the novel methods obtained higher sensitivities allowed reaching as low as 2.5–1.2% of the mutated DNA.The study demonstrates that early conventional methods could likely have underestimated the prevalence of KIT mutations of MCTs, therefore affecting the assessment of their relevance in prognosis and tyrosine kinase inhibitor (TKI) treatment effectiveness.     

Pulse pressure variation as a guide for volume expansion in dogs undergoing orthopedic surgery

Objective

To investigate whether pulse pressure variation (PPV) can predict fluid responsiveness in healthy dogs during clinical surgery.

A case-control study to identify farm factors affecting fertility of dairy herds: univariate description of factors

Objective To identify farm factors which were associated with reproductive performance in dairy herds in New South Wales.
Procedure A survey was administered by face to face interview to examine the responses of producers drawn from 757 herds, which used the New South Wales Agriculture Department Dairy Herd Improvement scheme. A case-control approach was used to select a total of 126 herds from the first (top group - cases) and fourth quartiles (low group - controls) for intercalving interval.
Results We found that the estimated interval from calving to first mating was significantly different between groups (P = 0.03) and that the groups significantly differed in both their target for interval to first mating (P = 0.02) and their perceived optimum time for first mating (P = 0.04). Other factors associated with a longer intercalving interval included, use of embryo transfer programs (P = 0.08), younger managers (P = 0.02), fewer breedings per day (P = 0.01), a greater number of people detecting heats (P = 0.07), but less hours spent detecting heats while handling the cows (P = 0.11), and a failure to vaccinate bulls for campylobacteriosis (P = 0.14).
Conclusions Managers of herds with poorer reproductive performance did not intend to mate cattle as soon after calving as managers with better reproductive performance, were not as active in seeking veterinary advice on reproduction, and were attempting to treat reproductive diseases and disorders themselves.     

Loading of VO2+ and Cu2+ to partially oxidized charcoal fines rejected from Brazilian metallurgical industry

Purpose

Charcoal is utilized in Brazil for the metallurgical industry. Small size pieces, called charcoal fines, are rejected and sometimes are used to produce energy by burning. This charcoal can be used as soil conditioner to improve retention of metal ions in soil. However, changing the charcoal's surface chemistry via oxidation may increase retention of metal ions.

Materials and methods

Two kinds of oxidants were employed, nitric acid and selenium dioxide/hydrogen peroxide. Vanadyl ion (VO²⁺) and copper ion (Cu²⁺) were utilized as probe to study the metal ion complexation by the partially oxidized charcoal obtained. FTIR and EPR spectroscopy were used to characterize the materials.

Results and discussion

Oxidation with nitric acid increased nitrogen content, while oxidation with SeO₂/H₂O₂ elevated carbon content of treated charcoals. Organic free radicals (OFRs) with the unpaired electron in p orbitals of aromatic structures were confirmed by the EPR g-factors that ranged from 2.0038 to 2.0031. The oxidation with selenium dioxide/hydrogen peroxide formed charcoal with largest quantity of OFR. Loading charcoal with VO²⁺ and Cu²⁺ resulted in formation of complexes were oxygen acted as coordination atom. The formed complexes were of axial symmetry. The configurations around the Cu²⁺ ions were: CUNCu²⁺ complex (more stable square planar symmetry) and CFNCu²⁺ complex (less stable distorted tetrahedral configuration). Both complexes had oxygen as coordinating atoms. CFNVO²⁺ and CFSeVO²⁺ complexes presented complexation sites of axial symmetry, C_4v, with oxygen as coordinating atoms.

Conclusions

The oxidative treatment of charcoal with SeO₂/H₂O₂ is more appropriate to produce soil organic conditioner for complexation of metal ions.     

The expression pattern of TIR8 is conserved among vertebrates

IL-1R/TLRs play an important role in the inflammatory responses. Their signaling pathway is tightly regulated to keep inflammation under control. The regulation involves both extracellular and intracellular mechanisms. TIR8, also known as SIGIRR (Single Immunoglobulin IL-1R-Related molecule), is an orphan receptor belonging to the IL-1R/TLRs super-family. Recent studies suggest its role in modulating the inflammatory response in the gastrointestinal tract in mice, acting as an IL-1 receptor family antagonist. We identified the sequence and analyzed the expression of TIR8 in a wide panel of organs and tissues in different animals. There was high homology of the cDNA sequence among human, mouse and the domestic species (74–85%). The pattern of expression of this receptor was similar in all the species examined (high levels in kidney and gastrointestinal tract) and similar to the human. These results demonstrate that TIR8 is conserved, in evolutionary terms, both with regard to sequence and pattern of expression, a finding consistent with a key function of this molecule in modulating inflammation in the gastrointestinal tract.     

Parameterization of ruminal fibre degradation in low-quality tropical forage using Michaelis–Menten kinetics

This work aimed to parameterize the ruminal degradation of neutral detergent fibre (NDF) from low-quality tropical forage using Michaelis–Menten kinetics. The intake, rumen outflow (L), fractional degradation rate (kd), discrete lag (LAG) and effective degradability (ED) of NDF, and the microbial flow of nitrogenous compounds into the small intestine (Nmic) were assessed in two 5 ×  5 Latin square experiments by using five Holstein × Zebu heifers cannulated in the rumen. The experiments were carried out sequentially and the treatments were formed by increasing the level of supplementation with nitrogenous compounds. A low-quality signal grass (Brachiaria decumbens) hay was used as roughage. The nitrogen supplement was a mixture of urea, ammonium sulfate and albumin, at the ratios of 4.5:0.5:1.0, respectively. The crude protein contents in the diets ranged from 51.9 to 136.3 g/kg of dry matter. The rumen ammonia nitrogen (RAN) concentration was used as an independent variable. The NDF intake, L and Nmic showed a quadratic pattern (P < 0.05) as a function of RAN concentration, and the critical points (maximum responses) were observed with 15.17, 16.28, and 14.52 mg of RAN/dL of rumen fluid, respectively. On the other hand, ED and LAG presented a linear-response-plateau (P < 0.05) according to the RAN concentration, with break points close to 8 mg/dL for ED (maximum estimate) and LAG (minimum estimate). The RAN concentrations to optimize NDF degradation and intake were defined as 8 and 15 mg/dL, respectively. This difference between estimates appears to be due to a better adequacy of the metabolizable protein:metabolizable energy ratio in the animal metabolism, which increases the animal intake even after the rumen NDF degradation has been optimized. This observation was supported by Nmic pattern. An adapted Michaelis–Menten model was applied to the data, where RAN was the independent variable and kd the dependent variable. The relationship between these variables was found to be significant by using the Hanes–Woolf plot (P < 0.01). Based on this model, the rate of NDF degradation as a function of RAN concentration indicates that fibre degradation in the rumen could be considered a second order process. In this context, the RAN concentration of 8 mg/dL was assumed as the limit where zero order (below limit) and first order (above limit) reactions become predominant for NDF degradation in the rumen.     

In vitro studies of Norwegian Red bovine semen immobilized and cryopreserved in alginate solid gel network

Development of new semen cryopreservation techniques improving sperm survival and ensuring availability of viable spermatozoa for a prolonged time‐period after AI is promising tools to reduce sensitivity of timing of AI and enhance overall fertility. The SpermVital^® technology utilizes immobilization of bull spermatozoa in a solid network of alginate gel prior to freezing, which will provide a gradual release of spermatozoa after AI. The objective of this study was to compare post‐thaw sperm quality and in vitro sperm survival over time of Norwegian Red bull semen processed by the SpermVital^® (SV) technology, the first commercialized production line of SpermVital^® (C) and by conventional procedure applying Biladyl^® extender (B). Post‐thaw sperm motility was not significantly different between SV, C and B semen (p > .05). However, sperm viability and acrosome intactness were higher for SV than C and B semen (p < .05). Small differences in DNA quality were observed (p < .05). Sperm viability after storage in uterus ex vivo was higher for SV than for C semen (p < .05). Furthermore, sperm survival in vitro over time at physiological temperature was significantly higher for SV semen than C semen as well as B semen during the incubation period of 48 hr (p < .05). In conclusion, the SpermVital^® technology is improved and is more efficient in conserving post‐thaw sperm quality and results in higher sperm viability over time in vitro for SV than for C and B semen.     

Dose-related antinociceptive effects of intravenous buprenorphine in cats

The dose-related antinociceptive effects of intravenous (IV) buprenorphine were evaluated in cats. Thermal (TT) and mechanical threshold (MT) devices were used for nociceptive stimulation. After baseline threshold recordings, buprenorphine was administered IV (0.01, 0.02 or 0.04 mg/kg; B1, B2 and B4, respectively) in a randomised, blinded and cross-over study. Data were analysed by ANOVA (P < 0.05) using 95% confidence intervals (CI). TT increased 15, 30, 45 min and 1 (5.2 ± 2.7 °C), 2, 3 and 4 h after B1; 15, 30, 45 min and 1 (5.1 ± 3.9 °C) and 2 h after B2, and 15, 30, 45 min and 1 (5.4 ± 3.3 °C), 2, 3, 6 and 8 h after B4. MT increased 15 and 45 min after B2 (260 ± 171 mmHg), and 30 (209 ± 116 mmHg) and 45 min and 1 and 2 h after B4. At 45 min, MT values were significantly higher after B2 compared to B1 (P < 0.05). With MT, B2 and B4 produced more antinociception and longer duration of action than B1, respectively. No dose response to thermal stimulation was detected.     

Central role for G protein-coupled phosphoinositide 3-kinase gamma in inflammation

Phosphoinositide 3-kinase (PI3K) activity is crucial for leukocyte function, but the roles of the four receptor-activated isoforms are unclear. Mice lacking heterotrimeric guanine nucleotide-binding protein (G protein)-coupled PI3Kgamma were viable and had fully differentiated neutrophils and macrophages. Chemoattractant-stimulated PI3Kgamma-/- neutrophils did not produce phosphatidylinositol 3,4,5-trisphosphate, did not activate protein kinase B, and displayed impaired respiratory burst and motility. Peritoneal PI3Kgamma-null macrophages showed a reduced migration toward a wide range of chemotactic stimuli and a severely defective accumulation in a septic peritonitis model. These results demonstrate that PI3Kgamma is a crucial signaling molecule required for macrophage accumulation in inflammation.