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

## TL;DR

Chilling boar sperm to 5°C does not impair intracellular calcium regulation after rewarming, which is important for fertility and assisted reproduction.

## Contribution

The study shows chilling boar sperm does not disrupt intracellular calcium store functionality after rewarming.

## Key findings

- Sperm stored at 17°C had higher initial intracellular Ca2+ levels than those stored at 5°C.
- Chilling did not affect Ca2+ release dynamics in viable sperm after rewarming.
- Thimerosal-induced Ca2+ responses were not significantly altered by storage temperature or duration.

## Abstract

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 (p < 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.