Analytical validation of five diagnostic tests for the detection of polymorphonuclear cells in stallion semen

The objectives of this study were to evaluate the ability of five 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 even low concentrations of PMNs affect motility. One ejaculate was obtained from six stallions. Aliquots of 50 × 10⁶ purified sperm were incubated, in triplicate, 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, 6) 10 × 10⁶ PMN/ml. The PMNs were quantified using a hemacytometer, cytology, a leucocyte esterase dipstick test (LEDT), a peroxidase test, and CD13 immunolabeling. Sperm motility was evaluated after 4 h at 38 °C. The number of leucocytes detected with the LEDT differed among treatments (P<0.0001), from negative results in control samples to moderate or large numbers in the samples with the highest PMN concentration. The hemacytometer count and CD13 immunostaining detected differences with the control treatment at the lowest PMN concentration (2.5 × 10⁶ PMN/ml; P<0.001). Sperm motion was lower in samples with ≥5 × 10⁶ PMN/ml (P<0.0001). Thus, a sample was considered leucospermic if it contained ≥5 × 10⁶ PMN/ml. The LEDT had the best sensitivity (100%), followed by cytology (78%), peroxidase test (60%), CD13 immunostaining (56%) and hemacytometer count (47%). The LEDT had the lowest specificity (65%), which was 95% for all other tests. In conclusion, the LEDT was a simple, economic and sensitive stall-side test to screen semen for presence of PMNs. Because of the lower specificity, positive LEDT results should be confirmed with the identification of peroxidase-positive cells or CD13-positive cells.