Antibiotic-free short-term storage of canine sperm at 5 °C preserves functional and mitochondrial integrity

The routine prophylactic use of antibiotics in sperm preservation for reproductive biotechnologies poses increasing concerns due to its potential role in promoting antimicrobial resistance. Therefore, developing storage systems that maintain sperm quality without antibiotics is essential. In dogs, chilled sperm is vital in assisted reproduction for genetic improvement and breed conservation. Typically, a 20% Tris–egg yolk extender with broad-spectrum antibiotics supports semen preservation for up to 3 days. This study evaluated a Tris–egg yolk medium without antibiotics, stored at 5 °C, as an alternative for short-term preservation of canine sperm. Eight ejaculates from dogs of different breeds were divided into two aliquots: a control group diluted in Tris–egg yolk with ampicillin, and a treatment group without antibiotics. Samples were stored at 5 °C and analyzed at 0, 24, and 48 h to assess motility, plasma membrane and acrosome integrity, intracellular oxidative stress, and mitochondrial membrane potential. No significant differences were found between treatments in most quality parameters during storage. However, storage time negatively affected membrane and acrosome integrity, as well as mitochondrial function, while slightly increasing oxidative stress. These results indicate that, under controlled short-term refrigeration conditions, canine sperm diluted in a Tris–egg yolk extender without antibiotics maintains functional parameters comparable to antibiotic-supplemented media for up to 48 h, despite time-dependent sub lethal cellular alterations.