Alfalfa silage has a high proportion of rumen-degradable protein content. Increasing dietary rumen-degradable starch (RDS) can enhance ruminal microbial protein synthesis. This study was conducted to investigate the influence of RDS levels in substrates with alfalfa silage on in vitro rumen fermentation and nitrogen (N) utilization. Rumen fluid was collected and dispensed into anaerobic fermentation bottles, each containing 1 g of substrate and 60 mL of rumen fluid–buffer mixture. The substrate was composed of 40% alfalfa silage and five different RDS levels: 14.85% RDS, 16.40% RDS, 18.67% RDS, 20.21% RDS, and 21.62% RDS. For each RDS level, three replicates were prepared. Each substrate was then incubated at 39 °C for 3, 6, 12, and 24 h. After incubation, the following parameters were measured: gas production, pH, α-amylase activity, ammonia nitrogen (NH₃-N), bacterial protein (BCP), and short-chain fatty acid (SCFA) concentrations were measured. Total gas production increased linearly with increasing RDS levels from 3 to 10 h of incubation (p < 0.01), with no difference observed among five levels after 11 h. At 3 h of incubation, pH decreased linearly with increasing RDS levels (p < 0.05). BCP concentrations and α-amylase activity increased linearly or quadratically with increasing RDS levels (p < 0.01), while the R4 group had the highest concentrations of BCP and the R5 group had the highest activity of α-amylase (p < 0.01). At 6 h of incubation, the NH₃-N concentration decreased linearly or quadratically with increasing RDS levels (p < 0.05), and the α-amylase activity, acetate, propionate, and total SCFA concentrations increased linearly (p < 0.01). The R4 group had the highest activity of α-amylase (p < 0.01), and the R5 group had the highest concentrations of acetate (p < 0.05) and propionate (p < 0.01). At 12 h of incubation, BCP, NH₃-N, and propionate concentrations, as well as α-amylase activity, increased linearly or quadratically with increasing RDS levels (p < 0.05). At 24 h of incubation, the α-amylase activity increased linearly with increasing RDS levels (p < 0.05). The highest multiple-factor associative effects index was observed in the 20.21% RDS substrate, indicating that an RDS level of 20.21% in the alfalfa silage substrate resulted in a desirable rumen N utilization.

Alfalfa forms and rumen degradable starch (RDS) levels in diets can profoundly affect growth performance and rumen fermentation patterns, this influence may result in variations in rumen microbiota. However, the effects of RDS levels on methanogenic and fungal communities in alfalfa hay (AH) or alfalfa silage (AS) diets, and the interaction between methanogens and fungi with growth performance and rumen fermentation patterns, remain unknown. In this study, a 2 × 2 factorial design resulted in four diets: two alfalfa forms (AH and AS) and two RDS levels (LR: 14.85% DM RDS; and HR: 20.21% DM RDS). We used 32 female Suffolk sheep for the experiment. On day 75 (including a 15-day transition period and a 60-day trial period), rumen content was collected after slaughter to examine the ruminal methanogens and fungi. The AHHR diet reduced the methanogen Chao 1 index compared to the AS diets (P < 0.05), and the Shannon index was lower than in the ASLR diet (P < 0.05). The fungi Chao 1 index was higher in the AH diets than in the ASHR diet (P < 0.05), and the fungi Shannon index was higher in the LR diets than in the HR diets (P < 0.05). The relative abundance of Aspergillus in the AHLR diet was significantly higher than in the AS diets (P < 0.01), and the relative abundance of Occultifur and Meyerozyma were decreased in the AH diets than in the AS diets (P < 0.05). The LEfSe analysis showed that Methanobrevibacter_sp_YE315 and Methanobrevibacter_sp_AbM4 were enriched in the ASLR diet, while Methanobrevibacter_millerae was enriched in the ASHR diet. For the fungal biomarkers, the AHLR diet included Aspergillus, Metschnikowia, and unclassified_f:Stachybotryaceae; the AHHR diet included stachybotrys, Stemphylium, and Cystobasidium; the ASLR diet included unclassified_k:Fungi, Trichothecium, and Psathyrella; and the ASHR diet included Alfaria. The correlation analysis results showed the relative abundance of Methanobrevibacter, Methanoculleus, Penicillium, Cladosporium, and Exophiala and the concentrations of isobutyrate and isovalerate, which may provide deeper insights into the previously observed differences.