Differences in the Gut Microbiota in Long-Term Infertile Holstein Repeat Breeder Cows and Healthy Fertile Holstein Cows

Repeat breeder (RB) cows are subfertile animals and represent a major issue in the livestock industry. The influence of the gut microbiota on reproductive function and the gut microbiota-gonadal axis has garnered scientific attention. This study aimed to evaluate the relationship between reproductive performance and gut microbiota in Holstein cows, depending on reproductive attempts (number of artificial inseminations or embryo transfers) and subsequent pregnancy outcomes. Fecal samples were collected from Holstein cows at the time of ovulation and classified into four groups: Normal pregnancy (<i>n</i> = 27, ≤three reproductive attempts, subsequently fertile), Normal open (<i>n</i> = 25, ≤three reproductive attempts, subsequently infertile), RB pregnancy (<i>n</i> = 38, over four reproductive attempts, subsequently fertile), and RB open (<i>n</i> = 60, over four reproductive attempts, subsequently infertile). The PLS-DA analysis revealed differences in classification among groups, especially between Normal pregnancy and RB open. Regarding α- and β-diversity analyses, these indices were significantly different among the four groups (<i>p</i> < 0.05). Using LEfSe analysis, three genus-level bacteria (<i>Bacillus</i>, <i>Ruminococcus</i>, and <i>Sphingobium</i>) were identified as specific bacteria in the Normal pregnancy group, and eight genus-level bacteria (<i>Huintestinicola</i>, <i>Intestinimonas</i>, <i>Neglecta</i>, <i>Oscillibacter</i>, <i>Zongyangia</i>, <i>Phocaeicola</i>, <i>Alistipes</i>, and <i>Acholeplasma</i>) were identified as specific bacteria in both open groups (<i>p</i> < 0.05). KEGG analysis predicted some pathways (Apoptosis, Lipopolysaccharide biosynthesis, etc.) to be enriched in both the open groups. MetaCyc analysis predicted amine and polyamine degradation/biosynthesis as the specific changing pathways in the RB open group. This study demonstrated that the composition of the gut microbiota differs between normal fertility and repeated infertility conditions. Furthermore, even with fewer reproductive attempts, differences in gut microbiota composition were observed between cows that conceived and those that failed to conceive.