2.8. Data Calculation and Statistical Analysis

Cumulative gas production data from the AGRS were fitted according to an exponential model as described by France [21]. In addition, the average gas production rate (AGPR) (mL/h) and hydrogen recovery (2Hrec) were also calculated following the description by Zhang et al. [22].

The data for gas production, IVDMD, fermentation gas composition, coenzyme contents, and VFAs were subjected to analysis of variance with the MIXED model procedure of SAS (Statistical Analysis for Windows, SAS Institute Inc., Cary, NC, USA). The model was applied as Y_ijk = μ + R_i + N_j + T_k + (N × T) _jk + e_ijk,, where Y_ijk is the dependent variable, μ represents the overall mean, R_i is the effect of the experimental run, N_j is the effect of the nitrocompound treatment, T_k is the effect of the incubation time, N × T are the interactions between the nitrocompounds and the incubation time, and e_ijk is the residual. The data for microbial abundance analysis were applied according to the model Y_ijk = μ + R_i + N_j + e_ijk, where Y_ijk is the dependent variable, μ represents the overall mean, R_i is the effect of the experimental run, N_j is the effect of the nitrocompound treatment, and e_ijk is the residual. Least square means and standard error (SEM) were calculated, and treatment differences were estimated using a multiple comparisons test (Tukey/Kramer). Correlation analyses between variables were performed using the CORR procedure of SAS. Significance was declared at p < 0.05.