Mitochondrial damage as an early event of monensin-induced cell injury in cultured fibroblasts L929

The present study was designed to identify, submicroscopically, the primary organelle or target structure for monensin in cultured murine fibroblasts L929. In addition, the effect of the drug on cell size and surface membranes of the cells were analysed; cellular proliferation, collagen secretion, and necrosis and apoptosis were re-evaluated. At the lowest concentration of monensin the foremost ultrastructural alteration occurred in the mitochondria, characterized by increased matrix density with disorganized and less distinct crystae. Incubation with monensin at higher concentrations resulted in severe mitochondrial damage and marked dilatation of the Golgi apparatus and rough endoplasmic reticulum cisternae. Fibroblasts exposed to higher concentrations of monensin were enlarged with decreased number of filopodia and hollows in the surface membrane. Moreover, monensin inhibited the cell proliferation, increased immunohistochemical positiveness for collagen type I in a dose-dependent manner, and, at high concentrations, caused cell necrosis whereas apoptosis was not induced. Taken together, these results show that monensin induces early mitochondrial damage, possibly causing an energy deficit that led to inhibition of fibroblasts proliferation and accumulation of collagen causing dilatation of Golgi apparatus and rough endoplasmic reticulum. Moreover, the mitochondrial damage would also explain the monensin-induced necrosis.     

The effect of different isolation procedures on canine leucocyte populations and on lectin-induced lymphocyte proliferation

Proliferation assays performed on peripheral blood mononuclear cells (PBMC) are commonly used in experimental and clinical immunology. A prerequisite for an in vitro assay is the ability to obtain relatively pure populations of mononuclear cells from whole blood, as contaminating polymorphonuclear cells may affect the proliferation of lymphocytes. Purification of canine leucocytes from whole blood is associated with difficulties in obtaining pure lymphocytes in high yields. The aim of this study was to optimize the lymphocyte purification from canine whole blood in terms of total cell recovery and purity, while not influencing the proliferation capacity of the isolated cells. To acquire optimal isolation of canine lymphocytes several density gradient media of different densities and osmolalities were examined. For optimal phagocyte removal, pre-treatment of whole blood with carbonyl iron/arabic gum and/or adherence to fibrinogen pre-coated polystyrene tissue flasks were examined. Lectin-induced proliferation was used as measurement of cell activity of the obtained cell fractions after the different separation procedures. Canine blood pre-treated with carbonyl iron/arabic gum followed by density gradient centrifugation with medium 'G' (density: 1.079 g/cm(3), osmolality: 256 mOsm) and adherence to pre-coated polystyrene tissue flask obtained the best PBMC cultures with a median lymphocyte purity of 88% and a median yield of recovered lymphocytes of 54%. This culture also resulted in the highest proliferation and subsequently the highest stimulation index upon lectin stimulation.     

Acral mutilation and analgesia in 13 French spaniels

Acral mutilation and analgesia (AMA) is reported in 13 French spaniels in Canada. This newly recognized disorder shares striking similarities in clinical features and biopsy findings to the other acral mutilation syndromes or hereditary sensory neuropathies reported in German short-haired pointer dogs, English pointer dogs and English springer spaniels. Clinical signs are first noted between 3.5 and 12 months of age. Affected dogs lick, bite and severely self-mutilate their distal extremities resulting in ulcers with secondary bacterial infection. Auto-amputation of claws, digits and footpads occurs in severe cases. Single or multiple feet can be affected. Affected dogs walked on their severely mutilated feet without evidence of pain, lameness, or ataxia. The majority of the dogs were euthanized within days to months of diagnosis.     

Germination of <Emphasis Type="Italic">Gibberella zeae</Emphasis> ascospores as affected by age of spores after discharge and environmental factors

The effects of age of ascospores (0–18 days after discharge), photon flux density (0–494 mol m^–2 s^–1 PAR), temperature (4–30 °C), frost (–15 °C for 30 min), relative humidity (RH; 0–100%), pH (2.5–6.5) and dryness (0 and 53% RH for up to 40 min) on the germination of the ascospores of the mycotoxin-producing fungus Gibberella zeae (anamorph Fusarium graminearum) were studied. Freshly discharged ascospores germinated within 4 h at 20 °C and 100% RH. The rate of germination and the percentage of viable ascospores decreased over time after the spores were discharged from perithecia. The time course of ascospore germination was not significantly affected by photon flux density. The period of time required to obtain 50% germinated ascospores at 100% RH was 26.90 h at 4 °C, 10.40 h at 14 °C, 3.44 h at 20 °C and 3.31 h at 30 °C. There was no significant effect of frost on the percentage of viable ascospores. A small percentage (6.6 ± 3.8%) of the ascospores germinated at 53% RH. At RH 84% and 20 °C almost 100% of the freshly discharged ascospores germinated. The time course of ascospore germination was affected by pH. The maximum rate of ascospore germination was estimated to be at pH 3.76. Ascospores lost their ability to germinate following exposure to 0% RH almost instantaneously. No germinating spores were detected after an incubation period of 1 min at 0% RH. Incubating the ascospores at 53% RH decreased the percentage of viable spores from 93 to 6% within 10 min. The data demonstrate that age of spores, relative humidity, temperature and pH, but not photon flux density, are key factors in germination of G. zeae ascospores.     

Unsaturated fatty acids from zoospores of <Emphasis Type="Italic">Sclerospora graminicola</Emphasis> induce resistance in pearl millet

Downy mildew of pearl millet, caused by Sclerospora graminicola, is a devastating disease, resulting in high economic losses in the semi-arid regions of the world. Recently, induction of host plant resistance using biotic and abiotic inducers are included among disease management practices as an eco-friendly approach. Unsaturated fatty acids are considered as a new generation of plant disease resistance inducers. In the present study, six unsaturated fatty acids, viz. docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), arachidonic acid (AA), linolenic acid, linoleic acid and oleic acid, all originally detected in the zoospores of S. graminicola,were applied to seeds of susceptible cultivars of pearl millet to examine their ability to protect against downy mildew under greenhouse and field conditions. In greenhouse experiments, EPA and AA induced a maximum of 78.6% and 76.5% protection, whereas linoleic acid, DHA and linolenic acid provided up to 66.3%, 61.2% and 24.5% protection, respectively. Oleic acid was not effective in protecting pearl millet (only 5.1% protection). A time interval of four days between treatment of seeds and challenge inoculation was required to obtain optimum protection. Plants raised from treated seeds and challenge inoculated at the tillering and inflorescence stages showed enhanced resistance, resulting in higher grain yield compared to untreated plants of the same cultivar. Chitinase activity was found to be higher in susceptible seedlings of pearl millet after treatment with the fatty acids and pathogen inoculation than in seedlings only inoculated with the pathogen. This indicates that host defence responses are activated and thus that induced resistance is involved in the protection observed. The role of unsaturated fatty acids as activators of resistance against downy mildew in pearl millet is discussed.     

Investigation of changes in the middle latency auditory evoked potential during anesthesia with sevoflurane in dogs

OBJECTIVE: To investigate the middle latency auditory evoked potential (MLAEP) in awake dogs and dogs anesthetized with 2 concentrations of sevoflurane. ANIMALS: 10 adult Beagles. PROCEDURE: The MLAEP was recorded while dogs were awake and anesthetized with sevoflurane (end-tidal concentration, 2.7% or 3.5%).Three needle electrodes were inserted SC, and click stimuli were delivered biaurally. Signal acquisition, averaging, and analysis were performed by use of computer software developed in-house. Signals were recorded for 128 milliseconds, and the responses to 1,024 stimuli were averaged. Waveforms from 10 recordings in each dog were averaged, and latencies of peaks were measured. Data acquired for awake dogs and dogs anesthetized with high and low sevoflurane concentrations were compared statistically. RESULTS: Sevoflurane anesthesia attenuated the MLAEP so that only peaks P0, Na, and Pa could be identified. The MLAEP changes were maximal at the lower concentration of sevoflurane evaluated. The latencies of these peaks were significantly shorter in awake dogs, compared with values in anesthetized dogs. No difference in the peak latency was detected between the sevoflurane concentrations. CONCLUSIONS AND CLINICAL RELEVANCE: In terms of CNS responsiveness, the effects of anesthesia with sevoflurane are similar to those of anesthesia with isoflurane. Data suggest that sevoflurane is not the inhalant agent of choice in a research setting where electroencephalographic measurements are to be recorded during anesthesia. The depression of the MLAEP waveform by sevoflurane also suggests that the MLAEP is not a suitable tool with which to monitor anesthetic depth during sevoflurane anesthesia in dogs.     

Biochemical analysis of the articular cartilage and subchondral and trabecular bone of the metacarpophalangeal joint of horses with early osteoarthritis

OBJECTIVE: To assess whether site-related changes in biochemical composition are present in the cartilage and subchondral and trabecular bone of the metacarpophalangeal joint of horses with early osteoarthritis. SAMPLE POPULATION: Right metacarpophalangeal joints from 59 mature warmblood horses. PROCEDURE: Biochemical data (cross-link, amino acid, DNA, and ash contents; denatured collagen and glycosaminoglycan [GAG] concentrations; bone mineral density; and mineral composition) were obtained from 2 differently loaded sites of phalanx I cartilage and subchondral and trabecular bone samples; data were compared with previously published values from nonosteoarthritic equine joints. RESULTS: Compared with findings in nonosteoarthritic joints, GAG concentration was lower in cartilage from osteoarthritic joints and there was a loss of site differences in cellularity and lysylpyridinoline (LP) cross-link content. In subchondral bone, LP cross-link content was decreased overall and there was a loss of site differences in osteoarthritic joints; ash content was higher in the osteoarthritic joints. Hydroxyproline content in trabecular bone from osteoarthritic joints was greater than that in nonosteoarthritic trabecular bone. In all 3 layers and at both sites, the linear increase of the pentosidine cross-link content with age had diminished or was not apparent in the horses with osteoarthritic joints. CONCLUSIONS AND CLINICAL RELEVANCE: In equine metacarpophalangeal joints with early osteoarthritis, distinct biochemical changes were detected in the cartilage and subchondral and trabecular bone. The dissimilarity in response of the different tissues and differences between the sites that are affected may be related to differences in biomechanical loading and transmission and dissipation of force.     

Reference range values of regional left ventricular myocardial velocities and time intervals assessed by tissue Doppler imaging in young nonsedated Maine Coon cats

OBJECTIVE: To describe and analyze the left ventricular free wall (LVFW) radial and longitudinal motions in a population of healthy Maine Coon cats by use of quantitative 2-dimensional color tissue Doppler imaging (TDI). ANIMALS: 23 healthy young Maine Coon cats (mean +/- SD: age, 2.1 +/- 0.9 years; weight, 5.0 +/- 1.0 kg). PROCEDURE: TDI was performed by the same trained observer (VC) on all cats. Radial LVFW velocities were recorded in endocardial and epicardial LVFW segments, and longitudinal velocities were recorded in the mitral annulus and in basal and apical LVFW segments. Isovolumic contraction and relaxation times were calculated in each myocardial segment, and the coefficients of variation (CVs; %) were determined for each TDI parameter. RESULTS: LVFW velocities were significantly higher in the endocardial layers than in the epicardial layers and also significantly higher in the basal than in the apical segments. Annular velocities were significantly higher than basal myocardial velocities in systole and early diastole. Coefficient of variation values were lower for radial velocities, particularly in systole, and were also lower for time intervals (16% to 22%) than for myocardial velocities (19% to 62%). CONCLUSIONS AND CLINICAL RELEVANCE: Because Maine Coon cats are predisposed to an inherited hypertrophic cardiomyopathy, which is a common cause of death in this breed, TDI could provide a useful tool for early detection of the disease. Tissue Doppler imaging indices may complete the conventional analysis of the left ventricular function in Maine Coon cats. However, the usefulness of TDI indices in the early detection of myocardial dysfunction needs to be clarified.     

Reproductive Disorders of Crossbred Dairy Cows in the Central Highlands of Ethiopia and Their Effect on Reproductive Performance

The study was conducted to estimate the proportion of reproductive disorders and to determine factors affecting reproductive performance of crossbred dairy cows under four different production systems in the central highlands of Ethiopia. The principal postpartum reproductive disorders were retained fetal membranes (14.7%) and uterine infection (15.5%). Anoestrus was the major postpartum reproductive problem in the mixed crop–livestock production system (38.6%) and was significantly associated with this production system. Apart from anoestrus, the occurrence of reproductive disorders was not significantly associated with a production system. Most of the reproductive disorders occurred as a complex rather than as a single abnormality. Two or more abnormal conditions were seen in 11.4% of the cases. Each reproductive trait measured was affected adversely by reproductive disorders. Cows with reproductive disorders in each production system, lactation group and suckling and non-suckling group had longer intervals from calving to first service and to conception (p < 0.001) and required more services per conception (p < 0.001). Pregnancy rate and conception to first service were 84.7% and 51.7%, respectively, for cows without reproductive health problems; and 64.2% and 15.1%, respectively, for cows with reproductive disorders (p < 0.001). Overall, intervals from calving to first service were shorter (p < 0.05) than in younger cows. Intervals from calving to first service and to conception were longer in suckling than in non-suckling cows (p > 0.05). Cows with a good body condition score (>3.5) at calving had shorter calving to first service and conception intervals than cows in poor condition (p < 0.001). The results showed that reproductive abnormalities, coupled with poor body condition, are important factors that contributed to reproductive inefficiency. An appropriate reproductive health management, a reliable artificial insemination service and supplementary feeding could be the management options to reduce or alleviate some of the problems.