The DM content of the feeds was determined by heating at 105 °C for 3 h (method 930.15; [21 ]), and the ash content was subsequently determined after incineration at 550 °C for 2 h (method 942.05; [21 ]). The neutral and acid detergent fiber (NDF and ADF, respectively) analysis was performed with a fiber analyzer (Ankom Technology, Macedon, NY, USA) following the procedure of Van Soest et al. [18 (link)] without correction for residual ash. The ether extract (EE) and the N contents were respectively determined by the solvent extraction and by the Kjeldahl methods (methods 954.02 and 976.05, [21 ]).
The pH and the NH3 content of the inoculum were measured with a glass electrode pH meter (GLP 22, Crison Instruments, S.A. Barcelona, Spain) and an ammonia electrode (Ammonia Gas Sensing Combination Electrode, ©Hach Company, Loveland, CO, USA, 2001).
For the VFA analysis, the aliquots of the inoculum were centrifuged at 20,000 g for 30 min at 20 °C and the supernatant was then filtered using polypore 0.45 µm filters (Alltech Italia, Milan, Italy). The filtrate was injected into a high-performance liquid chromatography instrument (Perkin-Elmer, Norwalk, CN, USA), set to 220 nm according to the method described by Martillotti and Puppo [22 ].
The pH and the NH3 content of the inoculum were measured with a glass electrode pH meter (GLP 22, Crison Instruments, S.A. Barcelona, Spain) and an ammonia electrode (Ammonia Gas Sensing Combination Electrode, ©Hach Company, Loveland, CO, USA, 2001).
For the VFA analysis, the aliquots of the inoculum were centrifuged at 20,000 g for 30 min at 20 °C and the supernatant was then filtered using polypore 0.45 µm filters (Alltech Italia, Milan, Italy). The filtrate was injected into a high-performance liquid chromatography instrument (Perkin-Elmer, Norwalk, CN, USA), set to 220 nm according to the method described by Martillotti and Puppo [22 ].