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Background:Hypothermic preservation of boar semen is considered a potential method for omitting antibiotics from insemination doses, thereby contributing to the global antibiotic resistance defence strategy. The main challenges are chilling injury to spermatozoa and bacterial growth during semen storage leading to reduced fertility. Objectives:To examine chilling injury and the number and type of bacteria in boar semen stored at 5 ℃ in the absence of antibiotics, and to assess the applicability of hypothermic semen storage under field conditions. Material and methods:Boar ejaculates were extended with AndroStar? Premium, stored at 17 ℃ with and at 5 ℃ without antibiotics and tested for functional sperm parameters by flow cytometry. Raw semen and extended samples were investigated bacteriologically. Fertility was evaluated after once-daily inseminations of 194 sows in a field study. Results:Lethal sperm damage assessed by motility and membrane integrity was low throughout storage in both experimental groups. Sublethal chilling effects based on the decrease of viable spermatozoa with low membrane fluidity were higher (P0.05) between sow groups inseminated with semen stored antibiotic-free at 5 ℃ and semen stored at 17 ℃ with antibiotics. Conclusion:Despite subtle chilling effects and low bacterial numbers, antibiotic-free hypothermic storage of boar semen offers the possibility to reduce the use of antibiotics in pig insemination. However, strict sanitary guidelines must be maintained and further evidence of efficiency under field conditions is considered desirable.