Recently, the importance of bacterial populations in various body niches has gained recognition in veterinary medicine, but research on the reproductive microbiome in dogs remains limited. Dog breeders consider the presence of bacteria in the genital tract as a threat to fertility, hence administration of antibiotics during mating or around parturition to improve fertility and puppy survival is common practice. Antibiotics are also routinely used in semen extenders, which are introduced into the genital tract of the bitch during artificial insemination. Moreover, first line veterinarians often prescribe antibiotics to bitches presented with vaginal discharge without further proof of a bacterial infection. During this research project we will investigate (1) the normal vaginal microbiome in healthy bitches, which will be linked with weight, age and parity of the bitch (2) the vaginal microbiome of bitches with vaginitis and (3) the effect of natural mating vs artificial insemination on the vaginal microbiome will be compared. Finally, we will examine possible alternatives to antibiotics in semen extenders. By means of next generation Nanopore sequencing techniques, this pioneering research will define the existence of the female canine reproductive microbiome under different circumstances, paving the way for evidence-based and reduced antibiotic use in canine reproduction.