## 4 Discussion  
The process of fermentation by ruminal cultures largely depends on available substrate. The pH of substrate fermentation is an important index to reflect whether rumen environment is healthy. In the present study, the pH values (6.4-6.8) were within the range of normality [27], which indicated that the rumen internal environment was static to some extent among different treatments. This likely due to the significant interaction effect on the pH of stage, treatment, and time. Moreover, the higher culture pH in the DWH7 group than in the MWS15 may be a result of the numerically higher ammonia concentration  
Figure 4: Bacterial community barplot analysis at the genus level in anaerobic fermentation of different treatments. (**a**) The relative abundance of the top 10 genera of rumen microbial community among treatments; (**b**) The differences of the genus _Ruminiclostridium_5_ among the treatments; (**c**) The differences of the genus _Methanobrevibacter_ between the groups. MWH7 = wheat harvested at 7:00 in milk stage and preserved as hay; MWS7 = wheat harvested at 7:00 in milk stage and preserved as silage; DWF7 = wheat harvested at 7:00 in dough stage and preserved as hay; DWS7 = wheat harvested at 7:00 in dough stage and preserved as silage; MWH15 = wheat harvested at 15:00 in milk stage and preserved as hay; MWS15 = wheat harvested at 15:00 in milk stage and preserved as silage; DWH15 = wheat harvested at 15:00 in dough stage and preserved as silage; DWS15 = wheat harvested at 15:00 in dough stage and preserved as silage. **_p_ < 0.01.  
in the DWH7 group than in the MWS15 group [28]. The gas production in vitro is a valuable index that describes the fermentability of ruminant feedstuffs and estimates DM degradation [29; 30]. The greater gas production in the wheat harvested at 15:00 (MWH15, MWS15, DWH15, DWS15) may because that the enzyme activity evaluated or some harmful bacteria were killed through the strong sunshine at noon, thereby increasing the digestibility of OM in the rumen fermentation. The higher DM degradation in the MWS15 group probably resulted from the greater microbial and enzymatic activity [31]. The higher aNDF degradation in the DWH7 group may be attributed to the more fibrolytic bacteria attached to the surface of the feed after the wheat was harvested at 7:00 in the dough stage and dried in the sun. In the process of rumen fermentation, there were more fibrolytic bacteria to be fond of digesting the wheat in the DWH7 group. Methane inhibition in the MWH7 group may be explained by the decreased availability of hydrogen for methanogenesis in the anaerobic fermentation of wheat hay [28]. The abundance of _Methanobrevibacter_ decreased significantly in the MWH7 group, while the methanogens have a symbiotic relationship with the protozoa in the rumen, which play a very important role in the generation of methane. The protozoa can produce a large number of hydrogen molecules in its own metabolic process, which can be used by methanogens to synthesize methane [32]. Therefore, we concluded that utilizing wheat hay in the ruminants not only benefits the global environment [33], but also reduces dietary gross energy loss [33]. CO\({}_{2}\) is one of the main gas composition in the anaerobic fermentation of rumen fluid, the higher CO\({}_{2}\) production in the wheat harvested at 15:00 probably because of the higher gas production in those groups, which resulted from the higher DM degradation [34]. Besides, CO\({}_{2}\) also is one of the main greenhouse gases in the atmosphere [35]. Therefore, the wheat group harvested at 7:00 (MWH7, MWS7, DWH7, DWS7) is beneficial for the global environment when fermenting in the rumen. Substrate composition affects the VFA concentration in the fermentation processes [36]. Consequently, the unaffected concentration of total VFA, acetate, and propionate, indicating that the fermentation pattern is similar, which could be explained by that all the substrate in the fermentation were wheat among different treatments.