Introduction: The aim of this study was to propose the optimal methodology for stallion semen morphology analysis while taking into consideration the staining method, the microscopic techniques, and the workload generated by a number of samples. Material and Methods: Ejaculates from eight pure-bred Arabian horses were tested microscopically for the incidence of morphological defects in the spermatozoa. Two different staining methods (eosin-nigrosin and eosin-gentian dye), two different techniques of microscopic analysis (1000× and 400× magnifications), and two sample sizes (200 and 500 spermatozoa) were used. Results: Well-formed spermatozoa and those with major and minor defects according to Blom’s classification were identified. The applied staining methods gave similar results and could be used in stallion sperm morphology analysis. However, the eosin-nigrosin method was more recommendable, because it allowed to limit the number of visible artefacts without hindering the identification of protoplasm drops and enables the differentiation of living and dead spermatozoa. Conclusion: The applied microscopic techniques proved to be equally efficacious. Therefore, it is practically possible to opt for the simpler and faster 400x technique of analysing sperm morphology to examine stallion semen. We also found that the number of spermatozoa clearly affects the results of sperm morphology evaluation. Reducing the number of spermatozoa from 500 to 200 causes a decrease in the percentage of spermatozoa identified as normal and an increase in the percentage of spermatozoa determined as morphologically defective.

Objective-To test horses for serologic evidence of an association between vesiviral antibodies and abortion. Sample Population-Sera from 141 horses. Procedures-2 experiments were conducted. Experiment 1 comprised sera obtained in 2001 and 2002 from 3 groups of horses (58 mares from farms with a history of abortion problems, 25 mares between 3 and 13 years of age with unknown reproductive histories that were sold at auction breeding-age control mares, and 29 mixed-age males and yearling females sold at auction negative control population). Experiment 2 comprised sera from 3 groups of pregnant mares (10 pregnant mares fed Eastern tent caterpillars ETCs, 9 pregnant mares fed ETC frass only, and 10 pregnant control mares). Sera were analyzed for antibodies against vesivirus by use of a validated recombinant vesivirus-specific peptide antigen in an indirect ELISA. Results-For experiment 1, 37 of 58 (63.8%) mares from farms with abortion problems were seropositive for vesivirus antibodies, whereas 10 of 25 (40%) breeding-age control mares were seropositive. All 29 mixed-age males and yearling females were seronegative for vesivirus antibodies. For experiment 2, 17 of 29 mares aborted (some from each group). Seropositive status for vesivirus antibodies increased from 47.1% (8/17) to 88.2% (15/17) for the pregnant mares that aborted during the experiment. Conclusion and Clinical Relevance-Significant association was detected between seropositive status for vesivirus and abortion in mares; consequently, vesivirus appears to be a pathogenic virus associated with abortion in mares. These data support adding vesivirus antibody testing into diagnostic screening to determine the cause for abortion in mares.

Six healthy adult horses (5 mares and 1 stallion) were given a single dose of acetylsalicylic acid (ASA), 20 mg/kg of body weight, by intravenous (IV), rectal, and intragastric (IG) routes. Serial blood samples were collected via jugular venipuncture over a 36-h period, and plasma ASA and salicylic acid (SA) concentrations were determined by high-performance liquid chromatography. After IV administration, the mean elimination rate constant of ASA (+/- the standard error of the mean) was 1.32 +/- 0.09 h−l, the mean elimination half-life was 0.53 +/- 0.04 h, the area under the plasma concentration-versus-time curve (AUC) was 2555 +/- 98 μg · min/mL, the plasma clearance was 472 +/- 18.9 mL/h/kg, and the volume of distribution at steady state was 0.22 +/- 0.01 L/kg. After rectal administration, the plasma concentration of ASA peaked at 5.05 +/- 0.80 μg/mL at 0.33 h, then decreased to undetectable levels by 4 h; the plasma concentration of SA peaked at 17.39 +/- 5.46 μg/mL at 2 h, then decreased to 1.92 +/- 0.25 μg/mL by 36 h. After rectal administration, the AUC for ASA was 439.4 +/- 94.55 μg · min/mL and the bioavailability was 0.17 +/- 0.037. After IG administration, the plasma concentration of ASA peaked at 1.26 +/- 0.10 μg/mL at 0.67 h, then declined to 0.37 +/- 0.37 μg/mL by 36 h; the plasma concentration of SA peaked at 23.90 +/- 4.94 μg/mL at 4 h and decreased to 0.85 +/- 0.31 μg/mL by 36 h. After IG administration, the AUC for ASA was 146.70 +/- 24.90 μg · min/mL and the bioavailability was 0.059 +/- 0.013. Administration of a single rectal dose of ASA of 20 mg/kg to horses results in higher peak plasma ASA concentrations and greater bioavailability than the same dose given IG. Plasma ASA concentrations after rectal administration should be sufficient to inhibit platelet thromboxane production, and doses lower than those suggested for IG administration may be adequate.

A quantitative real-time polymerase chain reaction method (qPCR) was developed and tested for the detection of Taylorella equigenitalis. It was shown to have an analytical sensitivity of 5 colony-forming units (CFU) of T. equigenitalis when applied to the testing of culture swabs that mimicked field samples, and a high analytical specificity in not reacting to 8 other commensal bacterial species associated with horses. As designed, it could also differentiate specifically between T. equigenitalis and T. asinigenitalis. The qPCR was compared to standard culture in a study that included 45 swab samples from 6 horses (1 stallion, 5 mares) naturally infected with T. equigenitalis in Canada, 39 swab samples from 5 naturally infected stallions in Germany, and 311 swab samples from 87 culture negative horses in Canada. When the comparison was conducted on an individual sample swab basis, the qPCR had a statistical sensitivity and specificity of 100% and 96.4%, respectively, and 100% and 99.1% when the comparison was conducted on a sample set basis. A comparison was also made on 203 sample swabs from the 5 German stallions taken over a span of 4 to 9 mo following antibiotic treatment. The qPCR was found to be highly sensitive and at least as good as culture in detecting the presence of T. equigenitalis in post-treatment samples. The work demonstrates that the qPCR assay described here can potentially be used to detect the presence of T. equigenitalis directly from submitted sample swabs taken from infected horses and also for determining T. equigenitalis freedom following treatment.

The object of this study was to evaluate recurrence of equine coital exanthema (ECE) whether re-infection or re-activation of causative virus. ECE is a venereal disease of horses caused by equine herpesvirus type 3 (EHV- 3). Like other herpesviruses, it may persist in infected horses for a long time. There is a controversy on the cause of ECE as the recurrence or the reinfection. This disease had occurred firstly on stallions and broodmares in Korea. The horses had rebreeded after healing routinely. Next year, the disease recurrented on the just same affected horses among stallions. The result of this study, re-outbreak of ECE in stallions is recurrence of ECE, but not reinfection of the virus.

A sample of testicular parenchymal tissue, approximately 2 X 7 X 7 mm, was aseptically removed from 1 testis in each of 9 stallions on day 0. Slight to moderate hemorrhage from the tunica albuginea was observed in 8 stallions, but bleeding from the parenchyma was detected in only 2 stallions. Stallions were castrated 27 days later. Normal development of granulation tissue was evident at the biopsy site, but hematomas were not observed. In situ measurement of the widths of the right and left testes, total scrotal width, and evaluation of testicular echogenicity during ultrasonography were variables used to monitor changes in the testicular parenchyma from 14 days before biopsy through 27 days after biopsy. The control testis was consistently larger than the biopsied testis, except for day 3. Ultrasonography revealed signs of a localized change in the parenchyma of the biopsied testis in 4 stallions, but each lesion decreased in size by day 27. Tissues removed during biopsy enabled an excellent appraisal of spermatogenesis at that time. Detailed examinations of seminiferous tubules in the testes were performed to assess for damage to testicular function. At castration, samples were taken from 6 sites in each testis. Quantitative histologic evaluations of testicular tissues revealed low numbers of spherical spermatids and pachytene spermatocytes in biopsied testes, compared with control testes. It was concluded that there was a transitory increase in degeneration of preleptotene spermatocytes and B spermatogonia at the time of biopsy. A mild inflammatory response at the biopsy site in some testes was evidenced by an increased number of leukocytes at the biopsy site and at a dorsal site. Because damage was minimal and appeared to be transitory, it was concluded that the open method of biopsy does not greatly alter the process of spermatogenesis or function of the testis in stallions.

Three ejaculates from each of 3 stallions were used to evaluate a computerized system (Hamilton-Thorn motility analyzer; HTMA) for measuring equine spermatozoal motility. Variance components (ejaculate-within-stallion, chamber-within-ejaculate, and microscopic field-within-chamber) were determined for each stallion after diluting ejaculates to 25 X 10(6) spermatozoa/ml with a skim milk-glucose seminal extender. The HTMA was compared with frame-by-frame playback videomicrography (VIDEO) for determining: percentage of spermatozoal motility and spermatozoal number in microscopic fields; curvilinear velocity and straight-line velocity of individual spermatozoa for 5 track types; and repeatability of those velocity measurements. The effect of spermatozoal number per microscopic field on incidence of intersecting spermatozoa and the outcome of intersecting spermatozoa also were evaluated. Greatest variability in motility measures was generally attributed to the microscopic field-within-chamber component. The HTMA was highly correlated with VIDEO for estimation of spermatozoal numbers per microscopic field (r = 0.99; P <0.001) and motility (r = 0.97; P <0.001); however over the entire range of spermatozoal numbers, the HTMA yielded higher spermatozoal numbers per microscopic field (P <0.05) and higher motility (P <0.05) than did VIDEO. The HTMA- and VIDEO-derived measurements of curvilinear and straight-line velocities were highly correlated for all spermatozoal track types, but both measures were higher (P <0.05) by use of the HTMA than by use of VIDEO for most track types. For 3 of 5 track types, measurements of curvilinear and straight-line velocities were less variable (P <0.05), using the HTMA, rather than VIDEO. Using the HTMA, the number of intersecting spermatozoa was highly correlated with spermatozoal numbers per microscopic field (r = 0.97; P <0.001). The percentage of erroneous track interpretations involving intersecting spermatozoa was high (85.3 +/- 2.7%). The HTMA was a reliable system for determining percentage of spermatozoal motility and velocity measures in video recordings of equine semen diluted to spermatozoal concentration of 25 X 10(6)/ml prior to evaluation.

Semen from three stallions was used to evaluate the effectiveness of two antibiotics added to semen extender for samples stored at 20 degrees C or 5 degrees C for up to 48 hours. Each ejaculate was divided into six different treatments: semen+extender (SE); SE+gentamicin (100 micrograms/mL); SE+polymyxin B (1000 units/mL); and each of the above treatments inoculated with Pseudomonas aeruginosa ATCC 27853. Sampling of diluted semen for bacteriological analysis was performed after 2, 8, 24 and 48 hours of preservation at either temperatures. The presence of nonspecific bacteria was noted after two hours in all SE aliquots. The number of bacteria did not change in samples stored at 5 degrees C, while in samples preserved at 20 degrees C, it increased by three to four times after 48 hours. In semen aliquots treated with either of the antibiotics, the number of nonspecific bacteria was very low after two and eight hours at both temperatures. This number remained stable up to 48 hours at 5 degrees C, while an increase was noted at 24 and 48 hours at 20 degrees C. At 5 degrees C, the number of P. aeruginosa cells tended to decrease between 24 and 48 hours in SE aliquots. The presence of gentamicin or polymyxin B appeared to rapidly inhibit growth of P. aeruginosa. At 20 degrees C, growth of P. aeruginosa increased between 8 and 24 hours in SE, while the presence of antibiotics almost completely inhibited the growth of the bacterium. In conclusion, gentamicin and polymyxin B appeared effective for the control of P. aeruginosa at either temperature, but nonspecific bacteria increased after 24 and 48 hours at 20°C.

Tissue variation in microscope slides made for spermatozoon analysis and variation introduced by the subjective techniques used to analyze these slides reduce the statistical power of studies that seek to use spermatozoon morphology to predict fertility. A simple specimen preparation method was developed to standardize stallion spermatozoon morphologic smears, and a new, automated spermatozoa morphometry instrument was used to objectively analyze the efficacy of the specimen preparation technique. The method achieved a standard spermatozoon concentration and reduced field-to-field variation in the number of spermatozoa analyzed. Metric measurements of spermatozoon head dimensions from clinically normal, fertile stallions revealed small, but highly significant, differences between stallions. The variation in metric measurements between replicate slides within stallions was small, indicating that replicate slide analysis probably is not necessary for clinically normal stallions. Coefficients of variation were generally less than 11% for metric measurements between stallions, and were less than 4% within stallions. This study revealed that a high degree of statistical power can be achieved when using these new, standardized specimen preparation and objective analysis techniques. Such power makes possible the detection of subtle differences between clinically normal stallions, and may facilitate accurate detection of abnormal fertility (ie, subfertility) in stallions.