Effect of N‐acetyl‐L‐cysteine Supplementation in Semen Extenders on Semen Quality and Reactive Oxygen Species of Chilled Canine Spermatozoa

The objective of this study was to evaluate the quality of chilled dog semen processed with extenders containing various concentrations of N‐acetyl‐L‐cysteine (NAC). Ejaculates from five dogs were collected, pooled and evaluated for concentration, motility, rapid steady forward movement (RSF‐movement), viability, acrosomal integrity and by the hypo‐osmotic swelling test (HOST). In addition, superoxide anion (O₂^‐?) production, hydroxyl radicals (OH^?) and total reactive oxygen species (tROS) were determined. The pool was divided into five aliquots, which were diluted to a final concentration of 66.66 × 10⁶ spermatozoa/ml with Tris‐glucose‐egg yolk extender containing one of the following concentrations of NAC (0, 0.5, 1, 2.5 or 5 mm ). The semen aliquots were chilled and preserved at 4°C. Semen quality was evaluated after rewarming at 72 h. Sperm motility was significantly higher with the 0.5 mm concentration compared with the control group (p = 0.001). Rapid steady forward movement was higher with the 0.5 and 1 mm concentrations compared with the control and 5 mm group (p < 0.001). Viability and HOST percentages were not significantly altered. Compared with the control, the 5 mm concentration showed significantly reduced percentages of spermatozoa with normal acrosomes (p = 0.049). None of the ROS values at 72 h were significantly affected by the presence of NAC in semen extenders, although all NAC concentrations showed lower O₂^‐? and OH^? values compared with the control. Only the concentrations of 1 and 5 mm inhibited the significant increase of tROS values after 72 h, compared with the fresh semen value. In conclusion, NAC supplementation of semen extenders is beneficial to semen motility of canine spermatozoa during chilling with the 0.5 mm concentration being the most effective, although no significant ROS inhibition was observed at 72 h.     

Effect of mitochondrial uncoupling and glycolysis inhibition on ram sperm functionality

Studies have demonstrated the importance of mitochondria to sperm functionality, as the main source of ATP for cellular homoeostasis and motility. However, the role of mitochondria on sperm metabolism is still controversial. Studies indicate that, for some species, glycolysis may be the main mechanism for sperm energy production. For ram sperm, such pathway is not clear. Thus, we evaluated ram sperm in response to mitochondrial uncoupling and glycolysis inhibition aiming to assess the importance of each pathway for sperm functionality. Statistical analysis was performed by the SAS System for Windows, using the General Linear Model Procedure. Data were tested for residue normality and variance homogeneity. A p < .05 was considered significant. Groups treated with the mitochondrial uncoupler Carbonyl cyanide 3 chlorophenylhydrazone (CCCP) showed a decrease in the percentage of cells with low mitochondrial activity and high mitochondrial membrane potential. We also observed that the highest CCCP concentration promotes a decrease in sperm susceptibility to lipid peroxidation. Regardless the lack of effect of CCCP on total motility, this substance induced significant alterations on sperm kinetics. Besides the interference of CCCP on spermatic movement patterns, it was also possible to observe such an effect in samples treated with the inhibitor of glycolysis (2‐deoxy‐d ‐glucose, DOG). Furthermore, treatment with DOG also led to a dose‐dependent increase in sperm susceptibility to lipid peroxidation. Based on our results, we suggest that the glycolysis appears to be as important as oxidative phosphorylation for ovine sperm kinetics as this mechanism is capable of maintaining full motility when most of the cells have a low mitochondrial membrane potential. Furthermore, we found that changes in the glycolytic pathway trough glycolysis inhibition are likely involved in mitochondrial dysfunction and sperm oxidative unbalance.     

Metaproterenol intoxication in a dog

Intoxication with metaproterenol, a mainly beta-2 selective agonist, was diagnosed in a dog with tachycardia, tachypnea, weakness, vomiting, and a history of exposure to the drug. Electrocardiography and echocardiography disclosed sinus tachycardia with episodes of ventricular tachycardia and exuberant systolic ventricular function, respectively. Administration of the beta blocking drugs propranolol and atenolol led to resolution of the clinical signs. Excessive sympathetic stimulation caused by metaproterenol is an unusual intoxication in dogs.     

Efficacy of digoxin for treatment of cats with dilated cardiomyopathy

The role of digoxin in treatment of cats with dilated cardiomyopathy and other forms of myocardial failure is unclear. We evaluated the chronotropic and inotropic effects of digoxin by comparing baseline, noninvasive indices of cardiac performance with those obtained after 9 +/- 1.3 (mean +/- SEM) days of digoxin treatment in 6 cats with heart failure attributable to dilated cardiomyopathy. Two-dimensionally directed, M-mode echocardiography and electrocardiography were used to determine left ventricular shortening fraction, preejection period (PEP), ejection time (LVET), PEP to LVET ratio, velocity of circumferential fiber shortening, electromechanical systole, heart rate, and PR interval. Treatment consisted of administration of furosemide (mean dosage, 2.4 mg/kg of body weight/day), digoxin in tablet form (approximately 0.01 mg/kg, q 48 h), aspirin (80 mg, q 48 h), and a commercial low-salt diet. In addition, 2 cats were administered short-term, low-dose fluids IV, and 2 were given taurine supplementation at rates of 500 and 1,000 mg/day. Other off-loading or inotropic agents were not administered. Therapeutic or toxic serum digoxin concentration was achieved in all cats. Significant (P less than 0.05) improvement was detected in mean values for shortening fraction, PEP, PEP to LVET ratio, and velocity of circumferential fiber shortening. Mean electromechanical systole and LVET did not change significantly. Improvement, as assessed by indices of cardiac function, was documented in 4 of the 6 cats treated with digoxin, including the 2 cats given taurine supplementation. In the cats given taurine, positive inotropic effect was observed prior to the time when taurine-induced improvement in ventricular function is detectable.(ABSTRACT TRUNCATED AT 250 WORDS)     

Strain differences in the susceptibility and resistance of Pasteurella multocida to phagocytosis and killing by rabbit polymorphonuclear neutrophils

The interactions of 2 capsular serotype A and 4 serotype D strains of Pasteurella multocida with rabbit polymorphonuclear neutrophils (PMN) were compared in vitro, using a PMN phagocytic and bactericidal assay. Bacteria and rabbit PMN were incubated for 15 minutes. The suspensions were subjected to differential centrifugation and the percentage of phagocytosis (cell association) was determined from the number of viable noncell-associated bacteria. The cell pellets and the associated bacteria were resuspended and PMN bactericidal activity was calculated from the number of remaining viable cell-associated bacteria at 45 and 75 minutes after the start of the assay. Test bacteria were not opsonized or were opsonized with immune serum containing active complement. One type A strain was ingested and killed by PMN in the presence and absence of opsonins. The 5 remaining strains were resistant to PMN killing, but only the type A strain resisted phagocytosis. Resistance of the type A strain was attributed to the hyaluronic acid capsule, since pretreatment of the bacteria with hyaluronidase rendered opsonized bacteria susceptible to ingestion and killing. The pattern of resistance of the 4 type D strains was different from that of the resistant type A strain. Both opsonized and nonopsonized type D bacteria became cell associated, but none were killed by PMN. The mechanism of resistance of these 4 strains to PMN bactericidal activity is currently unknown.     

Treatment of virulent footrot with lincomycin and spectinomycin

A mixture of lincomycin and spectinomycin was investigated as a treatment for footrot in sheep. In a controlled clinical trial 92.5% of acute and chronic cases of virulent footrot were cured following a single intramuscular injection of a mixture containing 50 mg lincomycin and 100 mg spectinomycin/ml at a dose rate of 1 ml/10 kg bodyweight. No improvement in clinical response was observed in groups of sheep treated on 3 successive days with this dose rate nor in another group treated once at a dose rate 1 ml/3.3 kg bodyweight. Cure effectiveness of each of the 3 treatment groups relative to untreated controls was 89%, 95% and 95%. Efficacy of lincomycin/spectinomycin was compared with that of penicillin/streptomycin in the treatment of footrot on 2 farms in south western New South Wales. Assessments made 14 to 17 d after treatment showed that on one farm all 122 ewes treated with lincomycin/spectinomycin had recovered while 170 of 175 ewes treated with penicillin/streptomycin recovered in the same period. On the second farm 87 of 90 ewes treated with lincomycin/spectinomycin recovered, compared with 184 of 190 sheep in the same flock treated with penicillin/streptomycin. Supportive footbathing did not seem to improve the clinical response in either treatment group and the paring done was sufficient only to establish diagnosis and to remove grossly overgrown horn.     

Primary left ventricular hemangiosarcoma diagnosed by endomyocardial biopsy in a dog

An 8-year-old spayed Golden Retriever was examined because of recent onset of congestive heart failure complicated by ventricular arrhythmias. Echocardiography revealed a thickened, hypokinetic region of the left ventricular free wall. Fluoroscopically guided transarterial endomyocardial biopsy of the hypokinetic region of the left ventricle revealed infiltration of the endocradium by neoplastic cells consistent with hemangiosarcoma. The dog's clinical condition deteriorated 7 weeks later, and necropsy confirmed the diagnosis and primary site of origin of the tumor. This report describes the clinical use of an endomyocardial biopsy technique to diagnose an infiltrating myocardial tumor that was associated with signs mimicking canine dilated cardiomyopathy.