Abstract
BACKGROUND: This study aimed to investigate differences in the structure and function of the rumen microbiome and its associated changes in rumen fermentation patterns and apparent nutrient digestibility in dairy cattle with different sorting behavior. Twenty-four Holstein cows in mid-lactation were initially enrolled in the experiment. All cows were fed and milked three times daily throughout the entire 28-day experimental period, comprising a 7-day pre-trial and a 21-day main trial. On days 1, 7, 14, and 21 of the main trial, feed sorting behavior was measured, and feed and feces samples were collected to determine apparent nutrient digestibility. Rumen content samples were collected on day 21 to measure pH, volatile fatty acids (VFA), and rumen microbiome structure and function. Based on feed sorting behavior, twelve cows were selected and divided into two groups: six cows that were severely sorted for fine particles-severely rejected long particles (SES; n = 6) and six cows that were slightly sorted for fine particles-slightly rejected long particles (SLS; n = 6). RESULTS: Comparative analysis revealed significant differences between the groups. The SES group exhibited lower rumen pH values and higher concentrations of total VFA (TVFA) and acetate (P < 0.05) than the SLS group. Data on apparent nutrient digestibility showed that compared to the SLS group, the SES group lowered the digestibility of neutral detergent fiber (NDF) and acid detergent fiber (ADF) (P < 0.05). Differential analysis of rumen microbiota indicated that the SES group had a higher relative abundance of Prevotella, Lactobacillus, Bifidobacterium, Selenomonas, and Acetitomaculum by a lower relative abundance of Fibrobacter, Ruminobacter, Pseudobutyrivibrio, Butyrivibrio, and Ruminococcus. Carbohydrate-active enzyme (CAZyme) annotation revealed that the SES group showed increased abundance of GH13 and GH65 enzymes, while exhibiting decreased abundance of GH1, GH3, GH5, GH6, and GH94. Functional profiling of Kyoto encyclopedia of genes and genomes (KEGG) modules revealed that compared to the SLS group, the rumen microbiota in the SES group upregulated the abundance of carbohydrate metabolism, amino acid metabolism, energy metabolism, and lipid metabolism. In carbohydrate metabolism, the rumen microbiota in the SES group upregulated the abundance of starch and sucrose metabolism, the citrate cycle, and pyruvate metabolism, while downregulating the pentose phosphate pathway. Functional profiling of KEGG Orthology (KO) enzymes revealed that the microbiota in the SES group preferred energy production through increasing glycolysis and supported the metabolism changes toward acetate production and fatty acid biosynthesis. CONCLUSION: Our findings reveal that feed sorting behavior significantly alters the rumen microbial ecosystem and its metabolic functions, negatively impacting fermentation efficiency, fiber digestibility, and overall nutrient utilization, even when cows are provided a well-balanced, standardized diet. This underscores the importance of early detection and management of feed sorting in dairy farms to promote cows' health and support sustainable dairy production.