Chilling Does Not Affect the Functionality of Intracellular Calcium Stores in Viable Boar Sperm During Liquid Preservation

In mammalian sperm, the regulation of intracellular calcium (Ca²⁺) is essential for fertility. Semen processing for assisted reproduction may disturb Ca²⁺ homeostasis. This study aimed to investigate whether chilling boar sperm to 5 °C and subsequent storage affect the function of intracellular Ca²⁺ stores. Semen was stored in BTS-extender at 5 °C or 17 °C (control) for up to five days. Fluo-4/AM-loaded aliquots were incubated in Ca²⁺-free Tyrode's medium at 38 °C. Sperm preserved at 17 °C had higher free intracellular Ca²⁺ levels compared with those stored at 5 °C (<i>p</i> < 0.05). However, there was no difference between storage groups in Ca²⁺ levels during incubation at 38 °C. Thimerosal, a sensitizer of Ca²⁺ channels, was added, and changes in the free intracellular Ca²⁺ concentration were monitored in viable acrosome-intact sperm by continuous flow cytometry. There was no effect of storage temperature on the kinetic response to thimerosal at days 1 and 3. At day 5, the relative increase in Ca²⁺ was higher in 5 °C-stored samples after 3 min of incubation. At 60 and 120 min of incubation, the thimerosal response was no longer influenced by the storage temperature or storage duration. In conclusion, chilling and storage do not affect the release dynamics of free Ca²⁺ from intracellular stores in viable boar sperm after rewarming.