Site-specific modulation of gut microbiota and fermentation patterns in lambs fed whole cottonseed

Background Whole cottonseed (WCS) is widely used in ruminant diets, but its impact on the spatial distribution of gastrointestinal microbiota and fermentation along the digestive tract remains unclear. Methods Twenty-four male lambs (Mongolian × Small-tailed Han sheep crossbred), aged 3-4 months and weighing ~30 kg, were randomly assigned to three treatment groups: control (CT, no cottonseed), 7.5% cottonseed (WCS7.5), and 15% cottonseed (WCS15). The experiment followed a completely randomized design with a 14-day adaptation period and a 60-day experimental period. Results Results showed that average daily gain and final body weights increased linearly with rising WCS inclusion ( P < 0.05), peaking in the WCS15 group. Dry matter intake remained consistent across groups, indicating feed conversion ratio (FCR) tended to decrease with WCS inclusion. At 30 days, WCS supplementation altered nutrient digestibility profiles, with WCS7.5 showing increased protein and fat digestibility. By 60 days, several digestibility parameters displayed quadratic responses, with greater values observed at WCS7.5 compared with WCS15, suggesting an inclusion-level-dependent response in nutrient utilization. Blood biochemical analysis revealed changes in serum biochemical profiles, including increased ALT and HDL cholesterol and decreased alkaline phosphatase (ALP) and gamma-glutamyl transferase (GGT) in the WCS15 group. Glucose and kidney function-related indicators were not adversely affected. Fermentation characteristics were altered, with WCS supplementation increasing the acetate-to-propionate ratio and producing site-specific changes in VFA profiles. Microbial diversity analysis showed limited overall effects of WCS supplementation; however, the observed feature count in the ileum was significantly affected by WCS inclusion (P < 0.05), indicating a site-specific response in microbial richness. Conclusion Overall, WCS supplementation resulted in site-specific alterations in gastrointestinal microbiota and fermentation characteristics, accompanied by changes in nutrient utilization and metabolic indicators. These findings suggest that the effects of WCS depend on both inclusion level and gastrointestinal location, emphasizing the importance of spatial microbial responses when evaluating dietary strategies in lambs.