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.

Epigenetic consequences of interploidal hybridisation in synthetic and natural interspecific potato hybrids.

Interploidal hybridisation can generate changes in plant chromosome numbers, which might exert effects additional to the expected due to genome merger per se (that is genetic, epigenetic and phenotypic novelties). Wild potatoes are suitable to address this question in an evolutionary context. To this end, we performed genetic (AFLP and single sequence repeart (SSR)), epigenetic (MSAP), and cytological comparisons in: (1) natural populations of the diploid cytotype of the hybrid taxonomic species Solanum × rechei (2n = 2×, 3×) and its parental species, the triploid cytotype of Solanum microdontum (2n = 2×, 3×) and Solanum kurtzianum (2n = 2×); and (2) newly synthesised intraploidal (2× × 2×) and interploidal (3× × 2×) S. microdontum × S. kurtzianum hybrids. Aneuploidy was detected in S. × rechei and the synthetic interploidal progeny; this phenomenon might have originated the significantly higher number of methylation changes observed in the interploidal vs the intraploidal hybrids. The wide epigenetic variability induced by interploidal hybridisation is consistent with the novel epigenetic pattern established in S. × rechei compared to its parental species in nature. These results suggest that aneuploid potato lineages can persist throughout the short term, and possibly medium term, and that differences in parental ploidy resulting in aneuploidy are an additional source of epigenetic variation.