Fig. 1: Characterization of normal and leukocytospermic semen of uninfected macaques.

a, b Representative immunocytochemical staining of cells from a normal semen (NS) and b leukocytospermic semen (LS). Numerous spermatozoa (black arrowhead) were present in NS. LS was rich in white blood cells, including lymphocytes (black *), macrophages (red arrowhead) and neutrophils (red *), frequently forming aggregates ([) with spermatozoa. c Gating strategy for semen leukocyte characterization. After the exclusion of doublets, cell debris, and dead cells, leukocytes are identified with the pan-leukocyte marker CD45. The SSC-A versus CD45 gate distinguishes lymphocytes from macrophages and polymorphonuclear cells on the basis of morphology. CD11b and HLA-DR distinguish HLA-DR^bright CD11b^{mid-to-bright} antigen-presenting cells (APC) from CD11b^bright HLA-DR^neg-to-low polymorphonuclear (PMN) cells. CD3⁺ T cells are gated against CD45 and CD4⁺ T cells are separated from CD8⁺ T cells. d–i Number of events acquired by flow cytometry per sample, as all collected semen cells were analyzed for NS (n = 7) and LS (n = 5): d CD45⁺ cells, e CD3⁺ lymphocytes, f CD4⁺ T cells, g CD8⁺ T cells, h APC, and i PMN. The dotted line in panels (d) represents the leukocytospermia threshold (10,000 CD45⁺ events acquired). The mean is shown. Statistical significance between groups was tested using Wilcoxon rank-sum tests, **p < 0.01.