Use of dimethylformamide to cryopreserve alpaca semen previously incubated with collagenase

The objective of this study was to evaluate the effect of collagenase and two final dimethylformamide (DMF) concentrations (4% and 7%) on alpaca frozen-thawed sperm quality. A total of 25 ejaculates from 5 alpaca were obtained using electroejaculation. Each individual ejaculate was evaluated and then diluted 4:1 in a solution of 1 mg/ml collagenase in HEPES-TALP medium and incubated for 4 min at 37°C. Subsequently, samples were diluted in TRIS-fructose-citric acid-egg yolk and cooled to 5°C. Then, each sample was divided in two aliquots and DMF at final concentration of 4% or 7% was added, equilibrated for 1 hr at 5°C and frozen over liquid nitrogen vapours. A Kruskal–Wallis test was used to evaluate the sperm morphometry, and Completely Random Block designs were used to analyse sperm motility, viability, membrane function and acrosome status. After collagenase incubation, none of the samples showed thread formation, and sperm parameters were preserved. Non-progressive motile sperm were higher (p < .05) in equilibrated samples (4% DMF: 31.8 ± 8.3% and 7% DMF: 36.3 ± 11.8%) compared to raw (10.1 ± 4.3%) and frozen-thawed semen (4% DMF: 9.7 ± 1.8% and 7% DMF: 7.5 ± 3.2%). Sperm membrane function, membrane integrity and intact acrosomes were higher (p < .05) in raw semen (40.1 ± 12.2%, 94.6 ± 3.2% and 91.3 ± 8.1%) compared to frozen-thawed samples (4% DMF: 19.8 ± 4.7%, 53.2 ± 2.7%, 65.7 ± 8.7% and 7% DMF: 20.4 ± 4.5%, 54.1 ± 1.4%, 64.6 ± 9.1%). Length of the sperm head was lower in frozen-thawed samples, being statistically different with 4% DMF compared to pre-freezing samples. The ratio between acrosome and head areas was greater (p < .05) in frozen-thawed samples. Incubation of raw alpaca semen with collagenase decreased the thread formation without affecting sperm quality. Frozen of collagenase treated alpaca semen with 4% or 7% DMF did not preserve the sperm parameters in thawed samples.     

Characterization of Mycobacterium avium subspecies paratuberculosis disseminated infection in dairy cattle and its association with antemortem test results

Mycobacterium avium subspecies paratuberculosis (MAP) disseminated infection in dairy cattle affects animal health and productivity and is also a potential public health concern. The study objectives were to characterize MAP disseminated infection in dairy cattle and to determine the role of antemortem tests in detecting cattle with disseminated infection. Forty culled dairy cows representing a variety of serum enzyme-linked immunosorbent assay (ELISA) results and body conditions were selected for the study. The physical condition of the cows was assessed via clinical examination prior to euthanasia and blood and feces were collected and tested by serum ELISA and fecal culture, respectively. Fifteen tissues were aseptically collected from each cow during necropsy and cultured for isolation of MAP. Disseminated infection was diagnosed when MAP was isolated in tissues other than the intestines or their associated lymph nodes (LNs) and was distinguished from infection found only in the gastrointestinal tissues and from absence of infection. Of the 40 cows in the study, 21 had MAP disseminated infection. Results showed that 57% (12/21) of cows with disseminated infection had average to heavy body condition and no diarrhea. Cows with disseminated infection had no to minimal gross pathologic evidence of infection in 37% (8/21) of cases. Only 76% (16/21) of cows with disseminated infection had positive historical ELISA results and only 62% (13/21) had a positive ELISA at slaughter. Thus, antemortem evidence of MAP infection was lacking in a high proportion of cows where MAP disseminated infection was confirmed.     

Thromboelastographic tracings in retired racing greyhounds and in non-greyhound dogs

BACKGROUND: Bleeding disorders in patients with normal coagulation test results are frequently reported in Greyhounds. The purpose of this study was to compare Greyhounds to non-Greyhounds by thromboelastography (TEG). HYPOTHESIS: TEG parameters in Greyhounds are different from those in non-Greyhounds. ANIMALS: Forty-three healthy dogs (28 Greyhounds and 15 non-Greyhounds) based on the results of physical examination, CBC, activated partial thromboplastin time, prothrombin time, fibrinogen, and platelet count. MATERIALS AND METHODS: Recalcified citrated native TEGs were performed in both groups; data were compared using Student's, Mann-Whitney, and Pearson's statistical tests. RESULTS: In Greyhounds, mean +/- SD were as follows: R-time 4.3 +/- 1.7 minutes, K-time 3.8 +/- 1.4 minutes, angle (alpha) 50.0 +/- 8.0 degrees , maximum amplitude (MA) 47.6 +/- 5.6 mm, clot strength (G) 4,647 +/- 1,097 dyn/cm2, and percent lysis at 60 minutes (LY60) 2.8 +/- 5.0%. In the non-Greyhounds they were R-time 3.7 +/- 1.6 minutes, K-time 2.5 +/- 0.9 minutes, angle 59.8 +/- 7.0 degrees , MA 53.1 +/- 5.6 mm, G 5,811 +/- 1,256 dyn/cm2, and LY60 3.1 +/- 2.5%. All parameters were significantly different between the groups, except for R-time and LY60. CONCLUSION: In Greyhounds, clotting kinetics are slower and clot strength are weaker than in non-Greyhounds, supporting the increased tendency to bleed observed after minor trauma or surgical procedures in the breed. The findings may also be attributed to blood viscosity or to the concentration of citrate in the sample (ie, Greyhounds have higher hematocrit and less plasma per unit volume).     

Renal handling of calcium and phosphorus in experimental renal hyperparathyroidism in dogs

Twenty-four hour urinary excretion, fractional excretion and the filtered load of calcium and phosphorus were monitored as hyperparathyroidism evolved in a model of progressive canine renal failure. Thirteen beagles of both sexes aged four and a half months were used. Nine of them were subjected to a renal damaging schedule (neomycine, 60 mg/kg/48 h, IM, 32 weeks) in order to induce chronic renal failure leading to secondary hyperparathyroidism (2HPT group). The remaining four were kept as the control group. The experiment was conducted over 32 weeks. Blood and 24 h urine were collected every four weeks. Calcium, phosphorus and creatinine were analyzed. Plasma parathormone and calcitonin were determined at weeks 0, 12, 24 and 32. The level of renal function in the 2HPT animals was reduced to 25% of that of the controls (endogenous creatinine clearance was 0.45 +/- 0.22 mL/min/kg as opposed to 1.81 +/- 0.54 mL/min/kg). Hyperparathyroidism was confirmed by a progressive increase in the levels of the parathyroid hormone. Calcitonin levels were not modified. A tendency to hypocalcaemia was observed, reaching statistically significant levels from the twenty-eighth week of the study, when hyperphosphataemia also became significant. Daily urinary excretion of calcium and phosphorus remained at values considered normal throughout the experiment with no alteration imputable to the impaired renal function. This is explained by the decrease in the filtered load of these elements (in both cases statistically significant from the 24th week on) being associated with an increase in their fractional excretion. Thus, calcium and phosphorus urinary excretion values could be maintained in a normal range up to the end of the experiment, showing that renal calcium handling in dogs with experimentally induced renal failure seems to differ from that observed in human patients.     

C-reactive protein measurement in canine saliva.

An established time-resolved immunofluorometric assay designed for measurement of C-reactive protein (CRP) in canine blood was evaluated and validated for use in canine saliva. C-reactive protein was measured in saliva specimens from 5 healthy dogs before and after the injection of casein and in 37 dogs with different disease conditions. The analytical and functional limits of detection were 0.000053 microg/ml and 0.0091 microg/ml, respectively, and intra- and interassay coefficients of variation ranged between 6.7-9.9% and 8.5-16.5%, respectively. A recovery experiment showed no significant disagreement between detected values and expected ones, and saliva CRP concentration was measured in a linear and proportional manner. A positive correlation was found between CRP levels obtained in saliva and serum samples in the experimental (R2 = 0.76) and clinical studies (R2 = 0.70). The assay was able to detect significant differences between salivary CRP levels in healthy dogs and dogs with inflammatory processes. These results suggest that saliva can be used for CRP measurement in dogs. The use of saliva presents the advantage of an easier and less stressful sampling method for the animals, which might be performed outside of hospital environments.     

Cardiopulmonary effects of desflurane in horses

OBJECTIVE: To determine the cardiopulmonary effects of desflurane (DES) in horses. ANIMALS: Six healthy adult horses, three males and three females, aged 9 +/- 4 (mean +/- SD) years and weighing 370 +/- 36 kg. MATERIALS AND METHODS: Anaesthesia was induced with an O2 (10 L minute(-1)) and DES mixture (vaporizer setting 18%). After oro-tracheal intubation, horses were positioned in right lateral recumbency. Anaesthesia was maintained with DES in O2 (20 mL kg(-1) minute(-1)) delivered through a large animal circle breathing system. The minimum alveolar concentration of DES (MAC(DES)) that prevented purposeful movement in response to 60 seconds of electrical stimulation of the oral mucous membranes was determined for each horse. The delivered concentration of DES was then increased to achieve end-tidal concentrations corresponding to 1.5 x MAC(DES), 1.75 x MAC(DES), and 2.0 x MAC(DES). Heart rate (HR), mean arterial blood pressure (MAP), respiratory rate (fr), tidal volume (VT), minute volume (VM) and core temperature were determined, and blood samples for arterial blood gas analysis taken at each DES concentration. All data were analysed by two-way anova for repeated measures and Fisher's test for multiple comparisons. A probability level of p < 0.05 was applied. RESULTS: Desflurane concentrations of 2.0 x MAC(DES) increased HR whereas lower concentrations did not. Mean arterial pressure was not affected by 1.0 x MAC(DES) 1.5 x MAC(DES) or 1.75 x MAC(DES), whereas it decreased at 2.0 x MAC(DES). All concentrations of DES examined significantly depressed fr, VT and VM. CONCLUSIONS AND CLINICAL RELEVANCE: Desflurane concentrations between 1.0 and 1.75 x MAC(DES) reduces fr and VM but does not affect HR or MAP in horses.     

An in vitro study of hematopoietic progenitor cells from peripheral blood of rabbits

The purpose of this study was to isolate and cultivate a subpopulation of pluripotent stem cells (PSCs) from the peripheral blood of rabbits, which are frequently used in veterinary research as an animal model. Pluripotent stem cells, as described in human beings, are fibroblast-like cells that exhibit a CD34 marker, specific from other hematopoietic stem cells. Commonly used human commercial media has been researched for culturing rabbit PSCs. These findings allow us to contemplate the direct application of this simple and standardized methodology in several areas of study, such as of the pharmacological effect of many drugs on hematopoietic cells, veterinary practice, and even the study of new strategies in cellular therapy for some human diseases.     

Evidence for a relationship between bovine erythrocyte lipid membrane peculiarities and immune pressure from ruminal ciliates

Erythrocytes of bovines and other ruminants have a strikingly anomalous phospholipid composition, with low or absent phosphatidylcholine (PC) together with high sphingomyelin (SM) content. Here, we report the presence in normal bovine serum of high levels of anti-phospholipid antibodies of IgM isotype against, PC and the phosphono analogue of phosphatidylethanolamine, aminoethylphosphonolipid (AEPL), normally produced by rumen ciliates. In contrast, no antibodies were detected against SM or N-acyl-phosphatidylethanolamine (NAPE), the major components of bovine erythrocytes. In addition, we found that exposure of the ciliate Tetrahymena thermophila to bovine serum results in rapid lysis, an effect that was inhibited by adsorption of the serum with SM/AEPL liposomes. Furthermore, incubation with bovine serum had a similar effect on freshly obtained ruminal ciliates, and the lytic activity was eliminated by pre-adsorption of the serum with SM/PE liposomes. The ruminant mode of life with its concomitant ciliate fauna is hereby linked to the peculiar conformation of bovine erythrocyte membranes. We propose that the unique phospholipid composition of bovine erythrocytes appears as an evolutionary adaptation to tolerate the lytic effects of anti-phospholipid antibodies generated against AEPL, a membrane component of the huge mass of ruminal ciliates, necessary commensals of this group of mammals.