Optimizing Rooster Semen Preservation: Effect of Oxygen Exposure, Sample Rotation, and HEPES Buffer Supplementation

This study aimed to investigate physical and biochemical strategies to optimize the preservation and fertilizing capacity of rooster semen during chilled storage and after artificial insemination (AI), respectively. Two semen extenders-0.9% sodium chloride (NaCl) and IGGKPh-were evaluated through two factorial experiments. In Experiment 1, a 2 × 2 factorial design was used to examine the effects of oxygen exposure (aerobic vs. reduced-oxygen conditions) and tube rotation (rotated vs. non-rotated) on semen quality during 24 h of storage at 5 °C. Sperm quality was evaluated based on progressive motility, viability, pH, and malondialdehyde (MDA) concentration. IGGKPh was significantly more effective than NaCl in preserving sperm function, maintaining motility above 70% and viability near 90%. Aerobic conditions and tube rotation improved motility but also increased MDA levels, indicating a tradeoff between enhanced metabolic activity and oxidative stress. Semen stored in NaCl lost its fertilizing capacity after 22 h, whereas IGGKPh under aerobic and rotated conditions resulted in significantly higher fertility rates (91.77%) compared with non-rotated samples. In Experiment 2, the effects of HEPES buffer supplementation (present vs. absent) and handling temperature (5 °C vs. 25 °C) were evaluated under simulated AI conditions. Semen extended in IGGKPh was stored at 5 °C for 22 h prior to handling, while NaCl samples were used immediately after dilution. Sperm quality was assessed at 0, 30, and 60 min of exposure. HEPES significantly reduced MDA levels and improved motility and viability in both extenders. Fertility rates were highest in the HEPES-supplemented groups, especially under chilled handling. In conclusion, optimal preservation of rooster semen requires a combination of metabolic support, adequate oxygen availability, gentle mixing, and pH stabilization. While IGGKPh was effective for storage up to 24 h, its performance was further enhanced by HEPES buffer. These findings offer practical recommendations for AI programs in poultry, particularly under field conditions where temperature fluctuations and delayed insemination are common.