Numerous studies have shown the presence of DNA lesions in human spermatozoa affecting sperm quality. However, the nature of this anomaly and its relationship with patient etiology are poorly understood since different mechanisms can be involved in the formation of these novel DNA configurations including the action of a seminal plasma nuclease activity. The objective of this study was to assess the capacity of seminal plasma for producing endogenous DNA cleavage using nuclei of peripheral blood leukocytes as external targets. For this purpose, we used seminal plasma from fertile males with normal semen parameters to produce DNA cleavage in a sample of leukocytes. Three different tests were performed to visualize DNA cleavage: (a) DNase activity detection, (b) DNA Breakage Detection-Fluorescence In Situ Hybridization (DBD-FISH), and (c) Two-dimensional comet assay (Two-tail comet assay). Our results demonstrate that: (i) the seminal plasma is able to cleave DNA compacted with histones in the leukocytes; (ii) this DNA cleavage can be associated with DNase activity and (iii) DNA damage mainly corresponds to single-strand DNA breaks. In conclusion, capacity of seminal plasma for producing DNA cleavage represents a solid contribution to expand the analysis of the standard seminal profile and could constitute a putative diagnostic tool for evaluating male infertility.Abbreviations: ALS: alkali labile sites; ART: Assisted Reproduction Technologies; DBD-FISH: DNA Breakage Detection-Fluorescence In Situ Hybridization; DNA: deoxyribonucleic acid; DSBs-DNA: double-strand DNA; FITC: Fluorescein IsoThioCyanate; GEDA: Gravity Enforced Diffusion Assays; PBS: phosphate-buffered saline; ROS: Reactive Oxigen Species; SSBs-DNA: single-strand DNA; SSC: saline-sodium citrate.

DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) enables detection and quantification of DNA breakage in the entire genome or within specific DNA sequences in single cells. We used this method to visualize and evaluate DNA damage in pigeon erythrocytes that were induced by elevated temperature and hydrogen peroxide. We also examined morphological changes in the cell nuclei. DBD-FISH demonstrated a significant increase of DNA damage in a temperature dependent manner, which resulted in nuclear abnormalities associated with apoptotic cells. These cells gave strong nuclear fluorescent signals that indicated cell death.