Hypothermic storage of boar semen provides the possibility to omit antibiotics from semen extenders so long as sperm quality is maintained and bacterial growth prevented. The objective of this study was to determine an optimal cooling-rate frame for boar semen preserved at 5°C in an antibiotic-free extender. Semen from eight boars extended in AndroStar® Premium was cooled from 30°C to 5°C using seven different cooling rates, ranging initially from 0.01 to 0.36°C min-1 and reaching 5°C between 2 h and 24 h after dilution. Sperm motility, membrane integrity, membrane fluidity, mitochondrial membrane potential and the response to the capacitation stimulus bicarbonate remained at a high level for 144 h at 5°C when the semen was initially cooled in a cooling-rate frame ranging from 0.01 to 0.09°C min‑1 in the temperature zone from 30 to 25°C, followed by 0.02 to 0.06°C min-1 to 10°C and 0.01 to 0.02°C min‑1 to the final storage temperature. A cooling rate of 0.07°C min-1 in the temperature zone from 30 to 10°C led to a reduced response to bicarbonate (P < 0.01) and fast cooling to 5°C within 1 h with a cooling rate of 0.31°C min-1 resulted in lower values (P > 0.05) of all sperm parameters. In a further experiment, slow cooling with a holding time of 6 h at 22°C induced after 6 h storage a temporary increase in Escherichia coli of 0.5 × 103 to 2.4 × 103 CFU mL-1 in the sperm-free inoculated extender. Overall, the load of mesophilic bacteria in the stored semen was below 6 × 103 CFU mL-1, a level that is not regarded as critical for sperm quality. In conclusion, appropriate cooling protocols were established for the antibiotic-free storage of boar semen at 5°C, allowing the application of hypothermic preservation in research and in artificial insemination.