Simple SummaryThis preliminary study aimed to assess the presence of bacteria in pregnancy tissues belonging to healthy canine and feline feto-maternal units at term. Specifically, it included bitches and queens undergoing elective cesarean sections and their first extracted fetus. The placental side of the endometrium, amniotic fluid, and meconium were sampled as tissue representative of the intra-uterine environment during pregnancy. Sampling and laboratory protocols were elaborated to contrast the possibility for contamination and included strict selection criteria (only elective cesarean sections, no recent treatments with antimicrobials), sterility during sample collection, and sampling and laboratory controls. Samples were processed using both culture and molecular techniques (16S rRNA bacterial gene sequencing). When positive, culture revealed the presence of bacteria that are common contaminants and sequencing yielded a very low bacterial load. A difference was highlighted between canine and feline samples, suggesting a possible contamination from the skin of the dam, although the small sample size prevents any definitive conclusion. This study suggests that healthy canine and feline fetuses might develop in the presence of low amounts of bacterial components, although future research should include stricter protocols to check for contamination and provide information on bacterial viability.AbstractAt present, there are no data on the presence of bacteria in healthy canine and feline pregnancies at term. Here, we investigated the uterine microbiome in bitches (n= 5) and queens (n= 3) undergoing elective cesarean section in two facilities. Samples included swabs from the endometrium, amniotic fluid, and meconium, and environmental swabs of the surgical tray as controls. Culture and 16S rRNA gene sequencing were used to investigate the presence of bacteria. Culture was positive for 34.3% of samples (uterusn= 3, amniotic fluidn= 2, meconiumn= 4, controlsn= 0), mostly with low growth of common contaminant bacteria. With sequencing techniques, the bacterial abundance was significantly lower than in environmental controls (p< 0.05). Sequencing results showed a species-specific pattern, and significant differences between canine and feline bacterial populations were found at order, family, and genus level. No differences were found in alpha and beta diversities between feto-maternal tissues and controls (p> 0.05). Dominant phyla were Bacteroidetes, Firmicutes, and Proteobacteria in different proportions based on tissue and species. Culture and sequencing results suggest that the bacterial biomass is very low in healthy canine and feline pregnancies at term, that bacteria likely originate from contamination from the dam’s skin, and that the presence of viable bacteria could not be confirmed most of the time.