3.1. Chemical Compositions and Microbial Populations of Ingredients and Pre-Ensiled TMR
High CP (~288 g kg
−1 DM) and EE (~140 g kg
−1 DM) content were observed in WHDG (
Table 1). Common vetch had high CP content (~188 g kg
−1 DM) and buffering capacity (~320 mEq kg
−1 DM). The high buffering capacity of common vetch could be attributed to the high content of crude protein and potassium, calcium, and magnesium salts of organic acids [
26]. High WSC (~139 g kg
−1 DM) and ADFom content (~253 g kg
−1 DM) were found in whole-crop oats. Hulless barley straw had high DM (~968 g kg
−1 FW) and aNDFom (~576 g kg
−1 DM) content. High DM (~904 g kg
−1 FW) and WSC (~110 g kg
−1 DM) content were observed in the mixed concentrate.
Quality silage could be obtained from raw material that have proper DM content (250~400 g kg
−1 DM), low buffering capacity, a high content of WSC, and an adequate LAB population to compete against the undesirable microorganisms before fermentation [
27]. Therefore, a high DM content (~555 g kg
−1 FW) was found in pre-ensiled TMR (
Table 2), which may inhibit the lactic acid fermentation. The moisture of raw material plays a critical role in affecting fermentation products because moisture is demanded by LAB for metabolic reactions and has a great influence on the initial level and transport of O
2 during ensiling [
28]. The high DM content of silage may limit the multiplication and metabolic activity of the indigenous LAB strains [
29]. Hence, it is necessary to apply some bacterial additives to accelerate the lactic acid fermentation and enhance the silage quality. In addition, a sufficient part of raw material should be WSC content because it is a critical factor influencing the fermentation quality of silage. In this study, the pre-ensiled TMR had a WSC content of 95.6 g kg
−1 DM, and this was higher than 60~70 g kg
−1 DM (considered as a theoretical requirement for making good silage), which may benefit the growth of desirable microbes, promote lactic acid fermentation, and ensure the successful fermentation [
1].
It is worth noting that high populations of yeasts (~6.64 log
10 cfu g
−1 FW) and molds (~5.81 log
10 cfu g
−1 FW) were found in pre-ensiled TMR. During ensiling, the growth of yeasts induces a large loss of DM and energy, which are principally fermented to ethanol, CO
2, and water under anaerobic condition [
30]. After exposure to air, yeasts are the main initiators of aerobic spoilage by consuming lactic acid and WSC, thus raising the pH and inner temperature of silage [
31]. Finally, molds completely deteriorate the quality of silage [
32]. For quality silage, the populations of yeasts and molds should not exceed 3~4 log
10 cfu g
−1 FW [
33], and higher numbers of yeasts and molds in raw material indicate a higher risk of aerobic deterioration after exposure to air. Hence, it is necessary to use some organic acid salts to improve the fermentation quality and aerobic stability of TMR silage. Consequently, we hypothesized the pre-ensiled TMR prepared with WHDG could be utilized to produce quality TMR silage with the help of organic acid salts and bacterial additives.
3.2. Fermentation Profile, Microbial and Chemical Compositions of TMR Silage
In the current study, the pH in all groups was higher than the critical value of 4.2 [
26]. However, silage with the higher DM content stabilized at a higher pH [
34]. Hence, after 95 days of ensiling, all the TMR silage was well preserved in the range of the pH values (4.32~4.51) (
Table 3). Greater (
p < 0.05) lactic acid (76.5~82.4 vs. 62.5~66.6 g kg
−1 DM) content and lower (
p < 0.05) pH values (4.32~4.33 vs. 4.42~4.51) were found in the LB and LAC groups compared to other groups. This study demonstrated the efficiency of LAB inoculants in accelerating lactic acid fermentation and converting WSC into organic acids, thus lowering pH. Moreover, the LAC group had higher (
p < 0.05) lactic acid content (82.4 vs. 76.5 g kg
−1 DM) than the LB group, which can be attributed to the different metabolic pathways of LAB strains. Holzer et al. [
35] reported that
L. casei belongs to facultative hetero-fermentative LAB and usually ferments hexoses homo-fermentatively into lactic acid, but, under special conditions, ferments hexoses hetero-fermentatively into lactic acid, CO
2, and ethanol (or acetic acid).
L. buchneri belongs to the obligate hetero-fermentative LAB and ferments hexoses to lactic acid, CO
2, and ethanol (or acetic acid), and has a lower efficiency than
L. casei in producing lactic acid. Hence, in the LAB inoculants of silage,
L. casei performed better than
L. buchneri in accumulating lactic acid during ensilage. Moreover, the SDA group had the highest (
p < 0.05) acetic acid (~31.3 g kg
−1 DM) content and the CAP group had the highest (
p < 0.05) propionic acid (~7.41 g kg
−1 DM) content among all groups, which may have resulted from the ionization of sodium diacetate and calcium propionate to acetic and propionic acids.
In silage, a good fermentation requires butyric acid less than 5 g kg
−1 DM [
36]. Herein, all the groups had less than 2.00 g kg
−1 DM of butyric acid, suggesting good fermentation. The NH
3-N in silage is an indicator of the degree of protein degradation, which impairs the nutritive value of forage. In well-preserved silage, the NH
3-N content should not exceed 100 g kg
−1 total N [
37]. In the present study, all the groups had less than 90.0 g kg
−1 total N of NH
3-N, indicating a good fermentation quality. The high DM could restrict the growth of enterobacteria and clostridia [
38], which might be responsible for the low butyric acid and NH
3-N content in all groups.
Compared to the DM content (555 g kg
−1 FW) of pre-ensiled TMR, the DM content (497~530 g kg
−1 FW) in all TMR silages consistently declined after ensiling. This decrease could be attributed to the fact that the easily degradable constituents (WSC) of silage were transformed into organic acids, ethanol, and CO
2 by microorganisms during fermentation; as a consequence, the WSC content declined in all TMR silages. In addition, the EE content is beneficial to the growth of undesirable bacteria during ensiling, which is an important factor affecting the silage quality. The National Research Council (NRC) recommends that the maximum EE level in the diet be 60~70 g kg
−1 DM because too much fat decreases the digestibility and passage rate of fiber, thereby accelerating the growth of undesirable bacteria and reducing rumen fermentation [
39,
40]. Thus, the EE content (73.9~75.5 vs. 83.2 g kg
−1 DM) in the CAP, POS, SDA, and LB groups were lower (
p < 0.05) than that of the control, indicating that these additives are effective in reducing EE content during ensiling.
Yeasts could metabolize WSC to produce CO
2 and ethanol during ensiling [
41]. The activity of yeasts was inhibited by organic acid salts and bacterial additives, as indicated by the lower (
p < 0.05) ethanol (10.9~16.9 vs. 26.3 g kg
−1 DM) content than the control. This finding may be because the acidic condition and ionized organic acids restrict the activity of yeasts [
5]. The calcium propionate, potassium sorbate, and sodium diacetate additives could ionize to produce these organic acids (primarily propionic, sorbic, and acetic acids) and salt ions, which have antibacterial properties and acidic characteristics [
42]. The undissociated molecules of these organic acids could pass through the plasma membrane and liberate protons to acidify the cytoplasm, thus limiting the growth and activity of microorganisms [
43].
It is interesting to note that similar (
p > 0.05) LAB populations were found among the LB (7.48 log
10 cfu g
−1 FW), LAC (7.47 log
10 cfu g
−1 FW), and control (7.91 log
10 cfu g
−1 FW) groups. Muck [
44] reasoned that the absence of an inoculant impacts the LAB number after fermentation through its effect on related factors that restrict its growth, such as nutrient shortage, or the accumulation of excretory products of the organism, resulting in insufficient conditions to cause a rapid decline in pH and to overcome the epiphytic population. Similar results in silage have been described by Ni et al. [
45], who found no significant difference in LAB populations between LAB-inoculated and control silage. Hence, in the selection process of inoculant strains, it is critical to consider the survival of the selected strains until the end of the ensiling process.
3.3. Aerobic Stability
The microorganisms linked with aerobic deterioration can increase pH and cause nutrient loss [
26]. In the experiment, the pH increased by 0.05, 0.11, 0.04, 0.05, 0.06, and 0.12 for the control, CAP, POS, SDA, LB, and LAC groups, respectively, during 14 days of aerobic exposure (
Table 4). This finding indicated that all the groups were steady during aerobic exposure, probably due to the sufficient acetic acid (13.0~31.3 g kg
−1 DM) content in TMR silage after air exposure. Studies have demonstrated that acetic acid could effectively restrict the growth and activity of undesirable microorganisms, thus enhancing the aerobic stability of silage [
46]. Moreover, the lactic acid content in all groups decreased to varying degrees, which may be correlated with the consumption of lactate-assimilating yeasts [
47].
On day 14, the SDA group had evidently (
p < 0.05) or numerically (
p > 0.05) lower aerobic bacteria (5.54 vs. 5.82~5.97 log
10 cfu g
−1 FW), yeast (4.55 vs. 4.59~6.46 log
10 cfu g
−1 FW) and mold (3.45 vs. 3.87~4.61 log
10 cfu g
−1 FW) populations than other groups (
Table 5). It indicated that SDA had a superior inhibitory effect on the aerobic bacteria, mold, and yeast populations than other treatments after air exposure. It was probably because sodium diacetate can ionize to a large amount of acetic acid (25.8~31.3 g kg
−1 DM) with antimicrobial property during aerobic exposure, and the un-dissociated acetic acid could get through the plasma membrane and affect the microbial metabolism [
48]. Hence, the growth and activity of harmful microbes were greatly limited in SDA treatment.
3.4. In Vitro Digestibility and Gas Production
The IVDMD is a critical parameter that reflects the rate of feed utilization in the rumen, and the digestion of DM principally contains WSC, protein, fiber content, and other substances [
49]. Herein, CAP, POS, and SDA treatments increased (
p < 0.05) IVDMD (54.8~57.5 vs. 48.4%) and IVNDFD (48.4~51.6 vs. 41.1%) compared to control (
Table 6), which may be attributed to the lower (
p < 0.05) aNDFom (385~428 vs. 443 g kg
−1 DM) content in CAP, POS, and SDA treatments. Lema et al. [
50] reported that a negative correlation existed between IVDMD and NDF, and similar results were also found by Xu et al. [
51]. There is no significant (
p > 0.05) difference in SCFA, ME, and NE
L among all groups. All the treated groups had evidently (
p < 0.05) or numerically (
p > 0.05) higher cumulative gas production at incubation time 72 h (GP
72) (173~223 vs. 168 mL) and potential gas production (174~227 vs. 169 mL) than the control, which may be attributed to the inhibition impacts on the growth and activity of harmful microorganisms, reducing nutrient loss and supplying adequate substrates and energy for microbial metabolism [
5].