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Background: The goal of this study was to assess the microbial ecology and diversity present in the uterus of post-partum dairy cows with and without metritis from 24 commercial California dairy farms using shotgun metagenomics. A set subset of 95 intrauterine swab samples, taken from a larger selection of 307 individual cow samples previously collected, were examined for α and β diversity and differential abundance associated with metritis. Cows within 21 days post-partum were categorized into one of three clinical groups during sample collection: control (CT, n = 32), defined as cows with either no vaginal discharge or a clear, non-purulent mucus vaginal discharge; metritis (MET, n = 33), defined as a cow with watery, red or brown colored, and fetid vaginal discharge; and purulent discharge cows (PUS, n = 31), defined as a non-fetid purulent or mucopurulent vaginal discharge., Results: All three clinical groups (CT, MET, and PUS) were highly diverse, with the top 12 most abundant genera accounting for 10.3%, 8.8%, and 10.1% of mean relative abundance, respectively. The α diversity indices revealed a lower diversity from samples collected from MET and PUS when compared to CT cows. PERMANOVA statistical testing revealed a significant difference (P adjusted < 0.01) in the diversity of genera between CT and MET samples (R2 = 0.112, P = 0.003) and a non-significant difference between MET and PUS samples (R2 = 0.036, P = 0.046). ANCOM-BC analysis revealed that from the top 12 most abundant genera, seven genera were increased in the natural log fold change (LFC) of abundance in MET when compared to CT samples: Bacteroides, Clostridium, Fusobacterium, Phocaeicola, Porphyromonas, Prevotella, and Streptococcus. Two genera, Dietzia and Microbacterium, were decreased in natural LFC of abundance when comparing MET (regardless of treatment) and CT, while no changes in natural LFC of abundance were observed for Escherichia, Histophilus, and Trueperella., Conclusions: The results presented here, are the current deepest shotgun metagenomic analyses conducted on the bovine uterine microbiome to date (mean of 256,425 genus-level reads per sample). Our findings support that uterine samples from cows without metritis (CT) had increased α-diversity but decreased β-diversity when compared to metritis or PUS cows, characteristic of dysbiosis. In summary, our findings highlight that MET cows have an increased abundance of Bacteroides, Porphyromonas, and Fusobacterium when compared to CT and PUS, and support the need for further studies to better understand their potential causal role in metritis pathogenesis., (© 2023. The Author(s).)