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Typically, sperm viability is assessed by flow cytometry (FC) with propidium iodide (PI) as the fluorescent stain. However, as PI emits in the red spectrum, it can overlap with other commonly used fluorochromes. Utilizing a blue viability stain, such as DAPI, for semen evaluation would free the red channel, enabling the use of additional fluorochromes and expanding analytical possibilities. This study aimed to evaluate the efficiency of DAPI as a viability stain for FC in three livestock species (goat, rabbit, and cattle), for which three experiments were conducted. First, the effect of incubation time on sperm viability rates was examined with DAPI and PI. Results indicated that incubation time did not affect the sperm permeability when DAPI and PI were employed. Second, the efficiency of DAPI as a viability indicator on different livestock species was studied. Viability rates measured with DAPI in goat, rabbit, and bull sperm samples showed significant correlation with PI measurements, showing values of 0.87, 0.86, and 0.61, respectively (\n                    <jats:italic>P<\/jats:italic>\n                    \u2009&lt;\u20090.01). Third, the effect of intrasample variation on viability rate was analyzed. When bovine sperm samples were stained simultaneously with DAPI and PI, the correlation between the two protocols was 0.83. In conclusion, DAPI is an alternative to PI for assessing sperm viability in livestock species, whilst also highlighting the importance of possible inter-species differences.\n                  <\/jats:p>","DOI":"10.1007\/s11259-025-10948-w","type":"journal-article","created":{"date-parts":[[2025,10,20]],"date-time":"2025-10-20T10:23:45Z","timestamp":1760955825000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Evaluating DAPI stain to assess sperm membrane integrity by flow cytometry in livestock species"],"prefix":"10.1007","volume":"49","author":[{"given":"Lydia","family":"Mart\u00ednez-Rodrigo","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sabrina","family":"Gacem","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maren","family":"Br\u00fcggemann","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sandra","family":"Pinto","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eva","family":"Moc\u00e9","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daniel","family":"Serrano-Jara","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"M. 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