Assessment of the cryopreservation of equine spermatozoa in the presence of enzyme scavengers and antioxidants

OBJECTIVE: To evaluate the effect of the addition of enzyme scavengers and antioxidants to the cryopreservation extender on characteristics of equine spermatozoa after freezing and thawing. SAMPLE POPULATION: 2 ejaculates collected from each of 5 stallions. PROCEDURE: Equine spermatozoa were cryopreserved in freezing extender alone (control samples) or with the addition of catalase (200 U/mL), superoxide dismutase (200 U/mL), reduced glutathione (10 mM), ascorbic acid (10 mM), alpha-tocopherol (25, 50, 100, or 500 microM or 1 mM), or the vehicle for alpha-tocopherol (0.5% ethanol). After thawing, spermatozoal motility was assessed via computer-assisted analysis and DNA fragmentation was assessed via the comet assay. Spermatozoal mitochondrial membrane potential, acrosomal integrity, and viability were determined by use of various specific staining techniques and flow cytometry. RESULTS: The addition of enzyme scavengers or antioxidants to cryopreservation extender did not improve spermatozoal motility, DNA fragmentation, acrosomal integrity, viability, or mitochondrial membrane potential after thawing. Superoxide dismutase increased DNA fragmentation, likely because of the additional oxidative stress caused by the generation of hydrogen peroxide by this enzyme. Interestingly, the addition of the vehicle for alpha-tocopherol resulted in a significant decrease in live acrosome-intact spermatozoa. CONCLUSIONS AND CLINICAL RELEVANCE: The addition of antioxidants to the cryopreservation extender did not improve the quality of equine spermatozoa after thawing, which suggests that the role of oxidative stress in cryopreservation-induced damage of equine spermatozoa requires further investigation. Our data suggest that solubilizing alpha-tocopherol in ethanol may affect spermatozoal viability; consequently, water-soluble analogues of alpha-tocopherol may be preferred for future investigations.     

Detection of papillomavirus-DNA in mesenchymal tumour cells and not in the hyperplastic epithelium of feline sarcoids

Abstract We examined 12 formalin-fixed paraffin-embedded feline skin tumours which had the histopathological features of fibropapillomas for the presence of papillomavirus (PV) DNA using touchdown polymerase chain recation (PCR), DNA sequencing and nonradioactive in situ hybridization. Nine of the tumours contained a 102-bp PCR product demonstrated using consensus PV primers that amplify a portion of the L1 gene. The nucleotide sequences are closely related, but not identical to that of ovine PV type 2, rabbit oral PV and reindeer PV. The deduced amino acid sequences had strong homologies with the major capsid protein L1 of deer PV, bovine papillomavirus (BPV) 1 and BPV 2, and European elk PV. Although PV antigens were not detected in any of the tumours by immunohistochemistry, PV DNA was demonstrated in individual mesenchymal cells or cell nests of 4/12 tumours by in situ hybridization. A nonproductive infection of mesenchymal fibroblast-like tumour cells with a papillomavirus would explain the lack of PV antigen expression and the absence of PV DNA in the hyperplastic epithelium. Because these tumours and their pathogenesis are similar to equine sarcoids, we suggest that they should be reclassified as 'feline sarcoids' instead of fibropapillomas.     

Computed tomographic evaluation of a bronchogenic cyst in a German shepherd dog

A German shepherd dog was referred for further evaluation of a cavitary pulmonary lesion. Computed tomography identified a well-defined rounded radiolucent area in the left cranial lung lobe in continuity with the bronchial lumen. These findings were consistent with a bronchogenic cyst.     

Canine bartonellosis: serological and molecular prevalence in Brazil and evidence of co-infection with Bartonella henselae and Bartonella vinsonii subsp. berkhoffii

The purpose of this study was to determine the serological and molecular prevalence of Bartonella spp. infection in a sick dog population from Brazil. At the S?o Paulo State University Veterinary Teaching Hospital in Botucatu, 198 consecutive dogs with clinicopathological abnormalities consistent with tick-borne infections were sampled. Antibodies to Bartonella henselae and Bartonella vinsonii subsp. berkhoffii were detected in 2.0% (4/197) and 1.5% (3/197) of the dogs, respectively. Using 16S-23S rRNA intergenic transcribed spacer (ITS) primers, Bartonella DNA was amplified from only 1/198 blood samples. Bartonella seroreactive and/or PCR positive blood samples (n=8) were inoculated into a liquid pre-enrichment growth medium (BAPGM) and subsequently sub-inoculated onto BAPGM/blood-agar plates. PCR targeting the ITS region, pap31 and rpoB genes amplified B. henselae from the blood and/or isolates of the PCR positive dog (ITS: DQ346666; pap31 gene: DQ351240; rpoB: EF196806). B. henselae and B. vinsonii subsp. berkhoffii (pap31: DQ906160; rpoB: EF196805) co-infection was found in one of the B. vinsonii subsp. berkhoffii seroreactive dogs. We conclude that dogs in this study population were infrequently exposed to or infected with a Bartonella species. The B. henselae and B. vinsonii subsp. berkhoffii strains identified in this study are genetically similar to strains isolated from septicemic cats, dogs, coyotes and human beings from other parts of the world. To our knowledge, these isolates provide the first Brazilian DNA sequences from these Bartonella species and the first evidence of Bartonella co-infection in dogs.     

Binding of ciprofloxacin labelled with technetium Tc 99m versus 99mTc-pertechnetate to a live and killed equine isolate of Escherichia coli

This paper describes a simple methodology for evaluating the bacterial binding of ciprofloxacin labelled with technetium Tc 99m. Using this methodology, the binding of 99mTc-ciprofloxacin by live Escherichia coli was compared with the binding of 99mTc-ciprofloxacin by killed E. coli and the binding of 99mTc-pertechnetate (99mTcO4-) by live E. coli. The antimicrobial effect of 99mTc-ciprofloxacin on E. coli was evaluated. Four groups were defined: live E. coli with 99mTc-ciprofloxacin, live E. coli with 99mTcO4 , killed E. coli with 99mTc-ciprofloxacin, and killed E. coli with 99mTcO4-. After 0, 2, and 4 h of incubation of 1 x 10(8) colony-forming units of E. coli suspended in 5 mL of sterile distilled water with 1.85 MBq of 99mTc-ciprofloxacin or 99mTcO4, 1 mL from each sample was centrifuged. The radioactivity of the bacterial pellet and that of the supernatant were measured separately, and the percentage of sample radioactivity attributable to bacterial binding was calculated. Of the 99mTc-ciprofloxacin, 3.6% to 5.9% was bound to live or killed E. coli; only 0.1% to 0.2% of the 99mTcO4- was bound to live E. coli (P < 0.0001). No significant difference in 99mTc-ciprofloxacin binding was found between live and killed E. coli (P = 0.887). An antimicrobial effect on E. coli was seen with 99mTc-ciprofloxacin: colony counts were reduced after 4 h. The small amount of 99mTc-ciprofloxacin binding and the lack of difference in binding between live and killed E. coli may limit the utility of this methodology in evaluating the presence of E. coli infection.