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

Abstract 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 [Ca 2+ ] 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 [Ca 2+ ] i decreased over time (1 h: 63 ± 16%, 96 h: 29 ± 18%; p < 0.05). The [Ca 2+ ] 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 impairs capacitation‐associated cell functions.