In vitro aging of stallion spermatozoa during prolonged storage at 5 °C

Artificial insemination with chilled stallion semen is hampered by a limited period of maximum fertility maintenance (24-48 h). This study used multiparametric flow cytometry to simultaneously measure reactive oxygen species (ROS) production, mitochondrial function or [Ca2+]i and plasma membrane fluidity in viable, acrosome-intact spermatozoa, with the aim of providing insight into changes in sperm function during storage at 5 °C. High proportions of viable and acrosome-intact spermatozoa (71±8%) remained after 96 h of storage demonstrating that the basic integrity of the cells was well preserved (n= 17 stallions). In addition, more than 90% of viable, acrosome-intact spermatozoa had active mitochondria and low intra-cellular or mitochondrial ROS levels. By contrast, the percentage of viable, acrosome-intact sperm with low plasma membrane fluidity and low [Ca2+]i decreased over time (1 h: 63±16%, 96 h: 29±18%; p <0.05).The [Ca2+]i in viable sperm rose 3.1-fold (p <0.05) over the 4 days, and fewer spermatozoa responded to bicarbonate stimulation (1 h: 46±17%, 96 h: 19±12%) with an increase in plasma membrane fluidity following prolonged storage. Overall, prolonged storage of stallion semen at 5 °C resulted in disturbed calcium homeostasis and increased plasma membrane fluidity. The decline in fertility of stallion semen during cooled-storage may therefore relate to aspects of in vitro aging (changes in plasma membrane fluidity and intracellular calcium) which impair capacitation-associated cell functions.