Diagnosis of leucospermia in the stallion

Abstract

The lack of standardized tests and interpretative criteria makes the diagnosis of stallion leucospermia difficult. Our objectives were to evaluate the ability of six diagnostic tests to detect polymorphonuclear cells (PMNs) in stallion semen, and to determine the concentration of PMNs that affects sperm motility. We hypothesized that all tests have diagnostic value, and that low concentrations of PMNs affect sperm motility. One ejaculate was obtained from six stallions. Aliquots of 50 ×106 purified sperm from each stallion were incubated with six concentrations of purified PMNs: 1) no PMNs, 2) 0.25 ×106 PMN/ml, 3) 0.5 ×106 PMN/ml, 4) 2.5 ×106 PMN/ml, 5) 5 ×106 PMN/ml, and 6) 10 ×106 PMN/ml. Each treatment was performed in triplicate. The PMNs were quantified using a hemacytometer (phase contrast microscopy), cytology (light microscopy), a commercial leucocyte esterase test (dipstick), a commercial peroxidase test (light microscopy), and immunolabeling of the neutrophil marker CD13 (flow cytometry). Sperm motility was evaluated after 4 h at 38 °C. The frequency of results obtained with the esterase test differed among treatments (P<0.0001). Negative or trace results were observed only with ≤ 0.5 ×106 PMNs/ml. A small number was observed in intermediate samples. In samples with ≥ 5 ×106 PMN/ml, only moderate or large results were obtained. Among quantitative tests, the hemacytometer count and flow cytometry detected differences with the control treatment at the lowest PMN concentration (2.5 ×106 PMN/ml; P<0.001). Total and progressive sperm motility were lower in samples with ≥5 ×106 PMN/ml than in those with lower concentrations (P<0.0001). Thus, a sample was considered leucospermic with ≥ 5 ×106 PMN/ml. The leucocyte esterase dipstick test had the best sensitivity (100%), followed by cytology (78%), peroxidase test (60%), flow cytometry (56%) and hemacytometer count (47%). The esterase test had the lowest specificity (65%), whereas the specificity of all other tests was 95%. In conclusion, presence of ≥5 ×106 PMNs/ml was associated with impaired sperm motility. The leucocyte esterase dipstick test is a simple, economic and sensitive stall-side test to screen semen for presence of PMNs in quantities capable of affecting sperm motility. Because of the lower specificity, we recommend confirming positive esterase results with the identification of peroxidase-positive cells under light microscopy, or CD13-positive cells using flow cytometry.