Fixative composition and sample storage conditions influence the proportion of morphologically normal bull sperm

Sperm morphology is a critical component of bull breeding soundness evaluations (BBSE), yet fixation and storage methods remain poorly standardised. This study examined how fixative composition and sample storage conditions influence bovine sperm morphology. Angus bull semen (n = 11) was fixed and assessed across three experiments using 1000x oil immersion differential interference contrast microscopy. Experiment 1 tested glutaraldehyde-based fixatives under isotonic (409 mOsm/kg) and hypertonic (1917 mOsm/kg) conditions, and 10% neutral buffered formalin (NBF) adjusted to acidic (3.94), neutral (6.98) and alkaline (10.05) conditions. Experiment 2 compared fixatives varying in pH, osmolality and composition (10% NBF with and without methanol, 5% NBF with methanol, formal saline, 3% NaCl and 1.25% glutaraldehyde in PBS). Experiment 3 evaluated the stability of these fixatives at 4°C, 21°C or 37°C, analysing samples after 7, 14 and 28 days of storage. Neutral and isotonic adjusted fixatives yielded the highest percentage of normal sperm. 5% NBF with methanol produced the greatest proportion of morphologically normal sperm, indicating chemical composition is equally critical to fixation success as pH and osmolality. 5% NBF also maintained high normal sperm percentage for up to 28 days at 4 or 21°C, whereas all fixatives showed marked deterioration in morphology when stored at 37 °C. Standardising BBSE protocols to adopt 5% NBF with methanol and storage at or below 21 °C would minimise artefactual distortion, improve inter-laboratory consistency and enhance the long-term reliability of morphology assessments in bulls. Future research should validate these protocols across broader genetics and under field conditions.