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This study used multiparametric flow cytometry to simultaneously measure reactive oxygen species (ROS) production, mitochondrial function or [Ca<jats:sup>2+<\/jats:sup>]<jats:sub>i<\/jats:sub>and plasma membrane fluidity in viable, acrosome\u2010intact spermatozoa, with the aim of providing insight into changes in sperm function during storage at 5\u00b0C. High proportions of viable and acrosome\u2010intact spermatozoa (71\u2009\u00b1\u20098%) remained after 96\u2009h of storage demonstrating that the basic integrity of the cells was well preserved (<jats:italic>n<\/jats:italic>\u00a0=\u00a017 stallions). In addition, more than 90% of viable, acrosome\u2010intact spermatozoa had active mitochondria and low intra\u2010cellular or mitochondrial ROS levels. By contrast, the percentage of viable, acrosome\u2010intact sperm with low plasma membrane fluidity and low [Ca<jats:sup>2+<\/jats:sup>]<jats:sub>i<\/jats:sub>decreased over time (1\u00a0h: 63\u2009\u00b1\u200916%, 96\u2009h: 29\u2009\u00b1\u200918%;<jats:italic>p<\/jats:italic>\u2009&lt;\u20090.05). The [Ca<jats:sup>2+<\/jats:sup>]<jats:sub>i<\/jats:sub>in viable sperm rose 3.1\u2010fold (<jats:italic>p<\/jats:italic>\u2009&lt;\u20090.05) over the 4\u2009days, and fewer spermatozoa responded to bicarbonate stimulation (1\u00a0h: 46\u2009\u00b1\u200917%, 96\u2009h: 19\u2009\u00b1\u200912%) with an increase in plasma membrane fluidity following prolonged storage. Overall, prolonged storage of stallion semen at 5\u00b0C resulted in disturbed calcium homeostasis and increased plasma membrane fluidity. The decline in fertility of stallion semen during cooled\u2010storage may therefore relate to aspects of in vitro aging (changes in plasma membrane fluidity and intracellular calcium) which impairs capacitation\u2010associated cell functions.<\/jats:p>","DOI":"10.1002\/cyto.a.24712","type":"journal-article","created":{"date-parts":[[2022,12,15]],"date-time":"2022-12-15T13:15:21Z","timestamp":1671110121000},"page":"479-491","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":18,"title":["In vitro aging of stallion spermatozoa during prolonged storage at 5\u00b0C"],"prefix":"10.1002","volume":"103","author":[{"given":"Muhammad","family":"Umair","sequence":"first","affiliation":[{"name":"Department of Clinical Sciences, Faculty of Veterinary Medicine Utrecht University Utrecht The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anthony","family":"Claes","sequence":"additional","affiliation":[{"name":"Department of Clinical Sciences, Faculty of Veterinary Medicine Utrecht University Utrecht The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marijn","family":"Buijtendorp","sequence":"additional","affiliation":[{"name":"Dierenartsenpraktijk Horst e.o. 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