4.1. Effects of Varying Proportions of Alfalfa Silage on the Fermentation Quality of TMR Silages
The high percentage of alfalfa silage may have directly produced an acidic environment in the AS60 silages. Therefore, the lactic acid content and pH in the AS60 group did not change significantly during ensiling, which is consistent with our previous findings [
5]. In contrast, the lactic acid contents in the AS40 and AS20 groups gradually increased during ensiling due to the lower original silage contents. The lactic acid contents of TMR silage in the AS40 and AS60 groups, which mainly originated from alfalfa silage, tended to decrease on the seventh day of ensiling. This result may be due to residual oxygen at the beginning of fermentation promoting the growth of aerobic microorganisms (such as molds, yeasts, and aerobic bacteria), which can use lactic acid and other volatile fatty acids for metabolism [
19]. Moreover, previous studies showed that re-ensiled silage may have a lower NH
3-N content than normal silage [
5,
19]. A similar result was found in the present study, in which the NH
3-N content tended to decrease after 7 days of ensiling. All TMR silages in the present study showed higher V-scores on the seventh day due to lower NH
3-N, acetic acid, and propionic acid contents.
Butyric acid was detected in the AS20 silages after 14 days of ensiling. In the experiment conducted by Wang et al. [
20], butyric acid was also detected in alfalfa silage on the 14th day. This may be due to the occurrence of clostridial fermentation [
21]. Silage clostridia have both saccharolytic and proteolytic properties [
22]. Clostridia not only use WSCs, lactic acid, and acetic acid as substrates to produce butyric acid but also decarboxylate free amino acids to produce amines and NH
3 [
22]. Clostridia generally thrive in low-sugar silages with high moisture contents (>70%), pH values (>4.6), temperatures (>30 °C), and buffering capacities [
23]. The contents of WSCs and DM are considered the key factors that decrease the pH and inhibit clostridia in silage [
22]. Researchers have found that the optimal DM content of TMR is 45–60% [
7]. Therefore, the TMR silages in the present study exhibited high DM contents, although all had low sugar contents (12–15 g·kg
−1 DM). A higher DM content could reduce the availability of inorganic ions to form a buffer system with the weak organic acids produced in the silage and facilitate the fermentation of silage with a low carbohydrate content [
24]. Hao et al. [
25] found little difference in fermentation quality between TMR silages with DM contents of 51.7% FM and 56.8% FM when the WSC content of the feedstock reached 62–65 g·kg
−1 DM, and no butyric acid was detected in any samples. Therefore, the production of butyric acid in the AS20 group might be due to the lack of sufficient WSCs for fermentation under slightly higher dry matter conditions (53.7% vs. 50.1% FM). On the other hand, the differences in the acidic environment and LAB population caused by different alfalfa silage contents might also have an important impact on the fermentation quality. Compared to the AS20 silages, the AS40 silages predominately showed lactic acid fermentation, although the initial WSC contents of the TMR were also low, which was likely due to the higher alfalfa silage content exerting a stronger inoculant-like effect.
The pH of the AS20 group did not continue to decrease after 14 days of ensiling, while the NH
3-N content greatly increased probably due to the proteolytic properties of clostridia, creating a high buffering capacity in these TMR silages [
26]. The pH decrease due to lactic acid fermentation also depends on the buffering capacity of the crop [
27]. A higher buffering capacity will prevent a pH decrease in silage, even if the silage has a high lactic acid level. Furthermore, the conversion of lactic acid to H
2, CO
2, and butyric acid by clostridia also leads to an increase in the silage pH [
21,
22]. A fermentation environment with a high pH will further promote the growth of clostridia. Clostridial spoilage in the AS20 group after 14 days of fermentation caused increases in NH
3-N and butyric acid contents, which led to a rapid decrease in the V-score. The V-scores of the AS40 and AS60 groups without clostridial fermentation remained stable in the later stages of anaerobic fermentation. Therefore, it may be necessary to apply additional LAB inoculants or fermentable substrates in the AS20 group to promote lactic acid fermentation and inhibit the occurrence of clostridial spoilage.
4.2. Effects of Varying Proportions of Alfalfa Silage on the In Vitro Degradability of TMR Silages
Digestibility can be used to evaluate the nutritional value and intake of animal feed [
8]. In addition, in vitro gas production is an indicator of feed digestibility [
28], which can be used to predict the metabolizable energy of TMR silage [
8]. Du et al. [
29] investigated the relationship between the chemical composition of forage and the ruminal degradation of nutrients, and the results showed that DMD and NDFD were positively correlated with the CP content and negatively correlated with the NDF and ADF contents. Several studies have investigated the relationship between the CP level and gas production, and inconsistent and conflicting results have been obtained [
30,
31,
32,
33]. However, the literature results on the relationship between NDF and gas production are relatively uniform and indicate a negative correlation [
32]. Although the improvements in the cumulative gas production and gas production rate in the AS60 silages were not significant, the AS60 silages with higher alfalfa silage contents showed significantly higher DMD than other TMR silages, which may be due to the higher CP contents and lower NDF contents. Therefore, in the present study, an increased percentage of alfalfa silage in TMR had a positive effect on the digestibility of the feed.
4.3. Effects of Varying Proportions of Alfalfa Silage on the Aerobic Stability of TMR Silages
Some small-scale family farms may take more than 5 days to consume a large TMR silage bale (more than 800 kg, FM). Therefore, it is necessary to ensure sufficient aerobic stability of TMR silages. Aerobic deterioration of silage involves the loss of sugars, the generation of heat, and the evolution of NH
3 and CO
2 [
34,
35]. Fungi and yeasts in particular usually play large roles in aerobic deterioration in silage [
36]. However, the yeast counts in the AS20 silages were always below 10
4 cfu·g
−1, and no molds were detected during the 7 days of aerobic exposure. Moreover, the temperature of the AS20 silages did not increase until the end of the test. This might be due to the presence of large amounts of butyric acid in the AS20 silages, which is more antimycotic than acetic acid and inhibited the growth of aerobic microorganisms [
37]. Danner et al. [
38] found that the aerobic stability of silage was significantly improved when it contained small amounts of butyric acid (>5 g·kg
−1 DM). Therefore, less lactic acid was metabolized by undesired microorganisms in the AS20 silages, which maintained stable pH, CP, and WSC levels on the seventh day of exposure. Although the antifungal properties of butyric acid could improve the aerobic stability of TMR silage, its presence could also cause a reduction in DM intake [
39] and increase the probability of ketosis within the herd [
23]. Due to the presence of butyric acid, the V-scores of the AS20 silages were always very low, even though they did not continue to decrease during aerobic exposure. In contrast, the AS40 and AS60 groups had significant decreases in V-scores only on the seventh day of the exposure test due to increased NH
3-N contents caused by proteolysis.
In the two treatment groups with higher alfalfa silage contents, the increased proportions of alfalfa silage caused increases in the acetic acid and propionic acid contents, and this result is similar to that of Wang et al. [
20,
40]. The growth of clostridia in the AS20 group before aerobic exposure may have caused an increase in the acetic acid content through redox reactions of amino acids or the breakdown of lactic acid [
22], resulting in a slightly higher acetic acid content than that of the AS40 group. Although acetic and propionic acid contents generally contribute to better aerobic stability, their levels were low in all treatment groups in the present study. In a study by Danner et al. [
38], the improvement of aerobic stability by acetic acid was very limited when the acetic acid content was below 15 g·kg
−1 DM. Although the AS60 group had a higher propionic acid content, its aerobic stability was not better than that of the AS40 group. Therefore, the difference in aerobic stability between the AS60 and AS40 groups was likely not caused by differences in the VFAs of the silage. After exposure to air, lactic acid (produced during ensiling or provided by alfalfa silage) and residual sugars are potential available substrates for the growth of aerobic microorganisms [
41]. The large proportion of alfalfa silage in the AS60 group directly provided a stable acidic environment and reduced the consumption of WSCs during ensiling. Although the AS40 group did not have a lower lactic acid content or higher protective volatile fatty acid (acetic acid, propionic acid, and butyric acid) contents, the AS40 silages preserved less residual WSCs than the AS60 silages after fermentation. Higher remaining concentrations of unfermented sugars facilitate aerobic microbial growth [
42], which may be the main reason for the lower aerobic stability of the AS60 group than the AS40 group. Furthermore, the AS40 silages showed significantly higher NH
3-N contents than the AS60 silages after 5 days of aerobic exposure. Kung et al. [
43] found that a higher ammonia content could significantly improve the aerobic stability of silage, which could also explain the higher aerobic stability of the AS40 group. Correspondingly, the molds were detected later in the AS40 group than in the AS60 group (3 days vs. 7 days). Therefore, it is optimal to develop a stable acidic environment during ensiling by adjusting the proportion of alfalfa silage in the TMR, which may help reduce potential substrates for aerobic microorganisms and enhance the aerobic stability of TMR silage.