Implications of different hypothermic preservation conditions on boar sperm quality during storage: Evaluation in sperm-rich fraction and whole ejaculate.

Low-temperature storage of boar semen is a promising alternative to antibiotics in artificial insemination (AI), helping reduce antimicrobial resistance. However, optimal handling protocols remain undefined, especially with the increasing use of semi-automatic collection systems that collect the whole ejaculate (WE) instead of just the sperm-rich fraction (SRF). This study assessed the impact of different temperature conditions on sperm functionality using both SRF and WE samples. Semen doses were refrigerated under four protocols: A) 15 °C for 4 days (15-group); B) 5 °C for 4 days (5-group); C) 5 °C for 2 days then 15 °C until day 4 (5/15-group); D) 15 °C for 2 days then 5 °C until day 4 (15/5-group). Sperm motility, kinematic parameters, plasma membrane and acrosome integrity, and mitochondrial activity were evaluated on days 0, 2, and 4. Thermal stress tests (heat and cold), sperm metabolism (OCR, and ECAR), and IVF outcomes (in WE samples) were assessed on day 4. Sperm quality was slightly affected by storage at 5 °C and by temperature shifts (5/15 °C and 15/5 °C), though values remained within optimal ranges. Thermal stress responses were similar across groups, and quality differences observed during storage disappeared after stress exposure. No significant differences in sperm metabolism were found. While the 15/5-group showed higher penetration rates, all treatments supported IVF. In conclusion, 5 °C storage, alone or combined with 15 °C, preserves sperm function supporting its practical application in AI regardless of ejaculate fraction.