C-reactive protein concentrations in canine acute pancreatitis

Objective: To determine if C‐reactive protein (CRP) concentration is elevated in spontaneously occurring canine acute pancreatitis (AP), and to measure changes in CRP during the course of hospitalization. Design: Prospective study. Setting: Tufts University School of Veterinary Medicine Foster Hospital for Small Animals. Animals: Sixteen client‐owned dogs with AP and 16 healthy controls. Interventions: Blood samples were obtained from the AP group on the day of diagnosis (Day 1), and on Days 3 and 5, unless the dog died or was discharged from the hospital. Blood was obtained from the control dogs once. Measurements and main results: Serum CRP was measured using a commercial immunoassay for each dog with AP and for healthy controls. Day 1 CRP concentrations were significantly higher in the AP group (56.1±12.7 μg/mL) compared with controls (2.8±1.3 μg/mL; P<0.001). For the 7 dogs that had samples collected on all 3 days, the mean CRP concentrations decreased significantly (P=0.043) over the 5 days of measurement. Of the 16 dogs with AP, 14 were discharged from the hospital and 2 were euthanized. Conclusions: Serum CRP concentrations were elevated in this group of 16 dogs with spontaneously occurring AP. In the 7 dogs that had measurements on all 3 days, the mean CRP concentration decreased from the day of diagnosis to the measurement made 5 days later.     

Spermatic and oxidative profile of domestic cat (Felis catus) epididymal sperm subjected to different cooling times (24, 48 and 72 hours)

Cooling stored epididymal samples for several days allows facilities to transport and process genetic material post‐mortem. Improvements to this practice allow the preservation of sperm from domestic cats, which are the ideal study model for wild felids. However, the modifications in spermatic features and the oxidative profile are not fully understood in cats. This information is necessary for the development of biotechniques, such as new extenders for cryopreservation. Therefore, the purpose of this study was to evaluate the spermatic and oxidative profile in samples from the epididymal cauda of domestic cats cooled at 5°C for 24, 48 and 72 hr. Spermatozoa were collected from the epididymis cauda. Evaluations consisted of computer‐assisted sperm analysis (CASA), plasma membrane integrity (eosin/nigrosin), acrosome integrity (fast green/rose bengal), sperm morphology, sperm DNA integrity (toluidine blue), mitochondrial activity (3′3 diaminobenzidine), activity of the antioxidant enzymes glutathione peroxidase (GPx) and superoxide dismutase (SOD), measurement of lipid peroxidation (TBARS) and protein oxidation. A decrease in sperm motility parameters was observed after 72 hr of cooling (i.e. total and progressive) with a higher percentage of minor (37.7 ± 6.3%) and total defects (53.4 ± 6.3%). Additionally, a decrease in high mitochondrial activity (Class I: 16.6 ± 2.2%) occurred after 72 hr. The decrease in motility rates after a long cooling time probably was caused by the increase in sperm abnormalities. A long cooling time causes cold shock and mitochondrial exhaustion, but there was no observed change with the oxidative stress condition. Therefore, cat epididymal sperm stored at 5°C appear to maintain a high quality for up to 48 hr of cooling time.     

A fast,low‐cost and efficient method for the diagnosis of sperm DNA fragmentation in several species

Sperm DNA fragmentation is a condition that interferes directly in the reproductive efficiency. Currently, there are several methods for assessing the sperm DNA integrity, such as Alkaline Comet, TUNEL and Sperm Chromatin Structure Assay. However, many of these techniques are laborious and require high‐precision equipment. Thus, the development of new techniques can optimize the evaluation of sperm DNA damage. Therefore, the aim of this study was to standardize the toluidine blue (TB) stain technique for the analysis of DNA fragmentation of dog, cat, bull, stallion and ram spermatozoa. For this purpose, we used six animals of each specie (n = 30), in reproductive age. Sperm was collected by different methods according to the particularities of each species, and such samples were divided into two aliquots: a sperm sample was kept at 5°C (considered as intact sperm DNA), and the remaining samples were submitted to the induction of DNA fragmentation by exposure to ultraviolet light for 4 hr. Samples were then mixed with the intact sample to obtain known and progressive proportions of sperm with fragmented DNA (0%, 25%, 50%, 75% and 100%). Semen smears were performed and subjected to staining with TB. Blue‐stained spermatozoa were considered to have DNA fragmentation. We observed high linear regression coefficients between the expected proportion of damaged DNA and the results of TB for dog, cat, ram, bull and stallion samples. In conclusion, TB stain was considered a fast and effective technique for the study of spermatozoa DNA in several species.     

TILLING and Ecotilling the Plant Kingdom

The modem crop scientist has a large amount of available nucleotide sequence information to identify genes of potential agronomic importance. Using reverse genetic approaches, specific genes can be disrupted, and hypotheses regarding gene function directly tested in vivo. Although a number of reverse genetic methods have been introduced, many are limited in application because they are organism-specific, expensive, transgenic, or only transiently disrupt gene function. However, traditional mutagenesis using chemical mutagens has been widely used as a forward genetics strategy to create many new crop plant varieties at relatively low cost. Mutagens such as ethyl methanesulphonate （EMS） cause stable point mutations and thus produce an allelic series of truncation and missense changes that can provide a range of phenotypes （Greene et al., 2003）. TILLING （targeting induced local lesions IN genomes） is a high-throughput reverse genetic strategy that combines traditional mutagenesis and SNP discovery methods （Colbert et al., 2001; McCallum et al., 2000）. To identify mutations, target regions of-l.5 kb are amplified with fluorescently labeled gene specific primers. Heteroduplexes are then formed between wild-type and mutant strands, mismatches are cleaved by incubation with a single-strand specific nuclease,     

Hormone-sensitive lipase: sequence, expression, and chromosomal localization to 19 cent-q13.3

Hormone-sensitive lipase, a key enzyme in fatty acid mobilization, overall energy homeostasis, and possibly steroidogenesis, is acutely controlled through reversible phosphorylation by catecholamines and insulin. The 757-amino acid sequence predicted from a cloned rat adipocyte complementary DNA showed no homology with any other known lipase or protein. The activity-controlling phosphorylation site was localized to Ser563 in a markedly hydrophilic domain, and a lipid-binding consensus site was tentatively identified. One or several messenger RNA species (3.3, 3.5, or 3.9 kilobases) were expressed in adipose and steroidogenic tissues and heart and skeletal muscle. The human hormone-sensitive lipase gene mapped to chromosome 19 cent-q13.3.     

In vitro pharmacologic effect of two endothelin-1 antagonists on equine colonic arteries and veins

OBJECTIVE: To evaluate the effectiveness of 2 potential endothelin (ET)-1 antagonists in blocking the contractile responses of equine colonic vessels to increasing concentrations of ET-1. SAMPLE POPULATION: Mesenteric vessels from 6 clinically healthy horses. PROCEDURE: Colonic vessels (arterial and venous rings) were placed in organ baths with oxygenated Tyrode solution at 37 C. Each was attached to a force transducer interfaced with a polygraph, and 2 g of tension was applied and equilibrated for 45 minutes. Then, B-1 (PD 142893) and B-2 (PD 145065) ET-1 antagonists were tested. One ring from each vessel type was used as a control for determining concentration-response relationships of ET-1 (10(-10) to 10(-6)M). Three rings of each vessel type were incubated with 3 concentrations of each antagonist (10(-7), 10(-6), and 10(-5) M) for 30 minutes before ET induced contractions were determined. The maximum contractile response and pA2 values were determined. RESULTS: Vessels contracted in a concentration-dependent manner to ET-1. Arteries responded slowly but reached greater contractions. Veins responded immediately with sustained contractions. Both antagonists inhibited contractions in a concentration-dependent manner with significant differences at 10(-6) and 10(-5)M for arteries and 10(-5) M for veins. Complete blockade of contractions was observed with B-2 (10(-5)M). The pA2 values for B-1 were 8.26 and 6.82 for arteries and veins, respectively, whereas they were 8.25 and 7.21 for B-2. CONCLUSION AND CLINICAL RELEVANCE: Both antagonists effectively blocked ET-1-induced contractions of equine colonic vessels. Because B-2 is water soluble and caused complete blockade at 10(-5) M, it appears to be the preferred antagonist.     

Efficacy of milbemycin oxime in the treatment of canine generalized demodicosis: a retrospective study of 99 dogs (1995-2000)

Ninety-nine dogs diagnosed with generalized demodicosis were treated with milbemycin oxime. Cases diagnosed before two years of age were classified as juvenile-onset (53%) and those diagnosed after two years of age as adult-onset (47%). Dogs were considered cleared of mites when none could be demonstrated in scrapings and cured if no relapse was seen for 12 months. Eighty-five per cent (84/99) were cured with milbemycin oxime used for 1-6 months (mean 2.3 months) at a dosage of 0.5-1.6 mg kg-1 body weight (mean 0.75 mg kg-1). No significant difference in dosage or treatment time was seen between juvenile and adult cases. Chance of cure was significantly better in young animals (cured cases mean age 2.97 years) than in older animals (not cured cases mean age 8.02 years). Start of treatment early in the course of disease gave a significantly better chance of cure. Cases with severe pododemodicosis had less chance of a cure (9/11 not cured).     

Agreement of high definition oscillometry with direct arterial blood pressure measurement at different blood pressure ranges in horses under general anaesthesia

ObjectiveTo determine the agreement of high definition oscillometry (HDO) with direct arterial blood pressure measurements in normotensive, hypotensive and hypertensive horses during general anaesthesia.Study designExperimental study.AnimalsSeven healthy warmblood horses, aged 3–11 years, weighing 470–565 kg.MethodsMeasurements from a HDO device with the cuff placed around the base of the tail were compared with pressures measured invasively from the facial artery. High blood pressures were induced by intravenous (IV) administration of dobutamine (5 μg kg⁻¹ minute⁻¹) over ten minutes followed by norepinephrine (0.1 mg kg⁻¹ IV) and low pressures by increasing the inspired fraction of isoflurane and administration of nitroglycerine (0.05 mg kg⁻¹ IV). For analysis three pressure levels were determined: high (MAP>110 mmHg), normal (60 mmHgResultsA total of 245 paired measurements of systolic (SAP), mean (MAP) and diastolic (DAP) pressures were obtained. The HDO device underestimated blood pressure at hypertensive and normotensive levels and overestimated blood pressure at hypotensive levels. Best agreement was obtained for SAP and MAP within normotensive limits. At normotension, bias ± standard deviation for SAP, MAP and DAP were 0.1 ± 19.4 mmHg, 0.5 ± 14.0, 4.7 ± 15.6, respectively. At high pressure levels bias and SD were 26.1 ± 37.3 (SAP), 4.2 ± 19.4 (MAP), 1.5 ± 16.8 (DAP) and at low pressures -20.0 ± 20.9 (SAP), -11.4 ± 19.6 (MAP), -4.7 ± 20.1 (DAP), with HDO measurements at a MAP <50 mmHg often failing.Conclusion and clinical relevanceGood agreement with invasive arterial blood pressures was obtained with HDO at normotensive levels in horses. At high and low pressure ranges HDO was unreliable. Therefore, if haemodynamic instability is expected, invasive measurement remains preferable.