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.     

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.     

Comparison of one-layer (continuous Lembert) versus two-layer (simple continuous/Cushing) hand-sewn end-to-end anastomosis in equine jejunum

OBJECTIVE: To evaluate single and double layer end-to-end anastomosis in equine jejunum. STUDY DESIGN: Experimental in vitro study. ANIMALS: Mid-jejunal sections from 12 adult horses without gastrointestinal disease. METHODS: Jejunal end-to-end anastomoses were performed by a continuous Lembert pattern or a simple continuous pattern oversewn with a Cushing pattern. Jejunal segments were distended with fluid at 1 L/min, and intraluminal pressure at failure, and mode of failure were recorded. Bursting pressure and bursting wall tension were calculated. Anastomosis construction time and degree of luminal reduction were recorded. Results- Single layer anastomoses were constructed in less time than 2-layer anastomoses. Both anastomotic techniques resulted in luminal reduction compared with control tissue; however, the reduction was smaller with a 1-layer continuous Lembert anastomosis. No differences were noted in bursting pressure or bursting wall tension between groups. CONCLUSIONS: Anastomosis using a 1-layer continuous Lembert pattern resulted in a larger stoma, was faster to perform, and as strong as a 2-layer anastomosis. CLINICAL RELEVANCE: Use of a 1-layer continuous Lembert pattern for jejunojejunosotomy may be beneficial by decreasing anastomosis time and produce a larger stoma than a 2-layer anastomosis.     

Hemorrhagic and necrotizing hepatitis associated with administration of a modified live canine adenovirus-2 vaccine in a maned wolf (Chrysocyon brachyurus)

A 15-yr-old, female, maned wolf (Chrysocyon brachyurus) was euthanized after presenting semicomatose with severe, uncontrolled frank hemorrhage from her rectum 6 days following a routine physical examination and vaccination. Histopathology indicated severe hemorrhagic and necrotizing hepatitis with intranuclear basophilic inclusion bodies in the liver that were thought to be consistent with adenoviral infection. Further classification by polymerase chain reaction, immunohistochemical staining, virus isolation, and electron microscopy confirmed the etiologic agent to be canine adenovirus-2. A representative sample of the vaccine that had been used was submitted and sequenced along with the virus isolated from the maned wolf. The sequencing of the etiologic agent that had been isolated from the maned wolf was determined to be the same as the strain of virus used in the production of the modified live vaccine that had been administered 6 days prior to death. From this information, the diagnosis of vaccine-induced adenoviral hepatitis was made. This is the first confirmed case of vaccine-induced canine adenoviral hepatitis in a maned wolf.     

Use of a three-drill-tract technique for arthrodesis of the distal tarsal joints in horses with distal tarsal osteoarthritis: 54 cases (1990-1999)

OBJECTIVE: To assess the long-term clinical outcome of horses with distal tarsal osteoarthritis (OA) in which a 3-drill-tract technique was used to induce arthrodesis of the affected joints, identify any preoperative or operative factors associated with outcome, and describe any complications associated with the technique. DESIGN: Retrospective study. ANIMALS: 54 horses. PROCEDURE: Medical records were reviewed for information on signalment, use, history, physical and lameness examination findings, surgical technique, and postoperative care. Radiographs were examined, and severity of OA was graded. Follow-up information was obtained through telephone interviews with owners at least 13 months after the procedure. RESULTS: 32 (59%) horses had a successful outcome, 6 (11%) improved but were not sound after surgery, and 16 (30%) did not improve following surgery. Outcome was negatively associated with the previous use of intra-articular injections. Few postoperative complications were evident. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that distal tarsal OA in horses can be successfully treated by means of distal tarsal arthrodesis with a 3-drill-tract technique. Horses with advanced distal tarsal OA are likely to have poorer outcomes, and the procedure will likely be of minimal benefit in horses with concomitant causes of hind limb lameness prior to surgery and in horses with preexisting proximal intertarsal joint disease.     

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.