Reducing sperm concentration is critical to limiting the oxidative stress challenge in liquid bull semen.

Because of the short breeding season, the use of liquid bull semen is a viable option in seasonal grass-based dairy systems such as Ireland. Currently in Ireland, liquid bull semen contains approximately 5 million sperm per insemination dose and is used within 2.5d of collection. The hypothesis of this study was that reducing the sperm number per insemination dose would enable bull sperm to be stored for longer. Semen was collected at a commercial AI center and diluted to 1 (T1), 2 (T2), 3 (T3), 4 (T4), and 5 (T5) million sperm per 0.25-mL dose in caprogen diluent. On d 0.25 (6 h postcollection), 1, 2, 3, 4, and 5 postcollection, viability, oxidative stress, and mitochondrial activity were assessed using flow cytometry and the fluorescent probes propidium iodide, CM-H2DCFDA, and rhodamine 123, respectively. On the same days, glucose consumption, total antioxidant capacity, and progressive linear motility were assessed. We observed an effect of day and treatment on sperm cell viability, with the highest percentage live found in T 0005 and the lowest in T 0025 on all days. Oxidative stress in live sperm increased with duration of storage and was affected by treatment, being highest in T 0025 and lowest in T 0005 on all days (d 5: 56.4±2.76% and 28.8±1.22%, respectively; mean ± SEM). Both the total antioxidant capacity and percentage of live sperm positive for rhodamine 123 were unaffected by treatment. The concentration of glucose in caprogen declined with time and was lowest in T 0025 and highest in T 0005 on d 5. In conclusion, higher concentrations of sperm have detrimental effects on sperm cell viability and increase oxidative stress but have no effect on the mitochondrial activity of sperm.