Replacing Dietary Roughage with Barley Hay Affects Rumen Fermentation, Microbial Community, Serum Immune and Antioxidant Status in Sheep

Barley hay (BH), characterized by high crude protein, neutral detergent fiber, and various bioactive compounds, is hypothesized to optimize ruminal microbiota and enhance systemic immune responses in sheep. This study assessed how the graded substitution of traditional forage with BH impacts ruminal fermentation, microbiota composition, and blood immune and antioxidant status in sheep. Forty-five male Dorper × Small-tailed Han crossbred lambs (aged 110 ± 10 days, BW 33.93 ± 1.11 kg) were stratified into five cohorts (n = 9 each), including a control (CON) and four experimental groups (BH25, BH50, BH75, and BH100), where BH replaced 25%, 50%, 75%, and 100% of their forage mixture, respectively. Results showed that BH supplementation consistently increased ruminal acetate concentrations. Specifically, BH75 and BH100 significantly enhanced the acetate-to-propionate ratio (p < 0.05) and concurrently reduced NH3-N concentrations in BH50 and BH100 (p < 0.05). Regarding ruminal microbiota, BH treatment did not alter alpha diversity but significantly enriched fiber-degrading bacteria, including Clostridia, Bacteroidales, and Prevotella, whereas the CON group favored Eubacterium. Blood analysis indicated a time-dependent modulation of immune and antioxidant markers. At day 30, BH treatment resulted in elevated immunoglobulin A (IgA), immunoglobulin G (IgG), interleukin-6 (IL-6), and malondialdehyde (MDA) levels (p < 0.05), whereas catalase (CAT) concentrations were markedly reduced (p < 0.05). By day 60, BH continued to enhance IgA, IgG, and IL-6 levels; meanwhile, tumor necrosis factor α (TNF-α) levels were up-regulated by BH25-BH75 (p < 0.05), while supplementation with BH75 and BH100 led to a decline in interleukin-4 (IL-4), CAT, and interleukin-10 (IL-10) (p < 0.05). Correlation analysis showed that a positive correlation was observed between total volatile fatty acids and Prevotella UCG-003/001, which conversely displayed a negative relationship with IgM. The norank f Eubacterium coprostanoligenes group showed an inverse association with valerate and IgG, but a positive correlation with interleukin-1β (IL-1β). In parallel, the norank f Bacteroidales RF16 group exhibited an inverse association with NH3-N and CAT, while unclassified c Clostridia was positively correlated with IL-1β. In conclusion, BH substitution modulates ruminal fermentation and immune responses by enriching fiber-degrading bacteria, though it may trigger oxidative stress. Given that BH100 maximized acetate and immunoglobulin production while minimizing MDA levels, complete replacement appears superior to partial substitution.