Calcium homeostasis role in preserving sperm function and metabolic activity during liquid storage of pig semen

Calcium (Ca²⁺) is known as a key regulator of sperm physiology, playing a crucial role in capacitation, hyperactivation, the acrosome reaction, and fertilisation. Despite this, whether it shapes the sperm's ability to withstand liquid preservation has not been addressed. Herein, we investigated how altering Ca²⁺ availability to pig sperm during storage at 17 °C affects their quality and metabolic activity. Using ethylene glycol tetraacetic acid (EGTA) and its membrane-permeable form (EGTA-AM), we selectively chelated either extracellular or intracellular Ca²⁺, respectively. Semen samples (n = 12 pools) were preserved in Beltsville Thawing Solution (BTS) under four conditions: control, Ca²⁺ supplementation, extracellular Ca²⁺ chelation, and intracellular Ca²⁺ chelation. Sperm function and metabolism were evaluated by flow cytometry and the oxygen consumption rate (OCR), whereas motility was assessed using Computer-Assisted Sperm Analysis (CASA). Ca²⁺ supplementation increased metabolic activity and induced capacitation-like changes, impairing sperm function. Intracellular Ca²⁺ chelation caused a sharp decline in motility and disrupted the metabolic balance, likely due to proton (H⁺) accumulation in the intermembrane space of mitochondria. In contrast, extracellular Ca²⁺ chelation preserved sperm viability, membrane integrity, and motility, while slightly reducing the OCR and the production of intracellular reactive oxygen species. These findings highlight the critical role of Ca²⁺ homeostasis in maintaining sperm function and suggest that fine-tuning Ca²⁺ dynamics - rather than full depletion - may improve the longevity and fertilising potential of liquid-stored pig semen.