1. Introduction
Soybean (
Glycine max) is currently one of the most important crops worldwide [
1], and soybean meal, as its coproduct has been considered the most economical plant protein in animal diets. Whole-plant soybeans (WPSs, including stems, leaves, and fruits) are also easy to produce on a large scale. However, WPS use in animal feeding has faced logistical and nutritional challenges.
Ensiling is commonly used to store forage biomass and maintain nutritional value by fermentation and acid production [
2]. After the aerobic stage of ensiling, anaerobic bacteria (mainly lactic acid bacteria—LAB) produce organic acids, reduce silage pH, and inhibit the growth of spoilage microorganisms [
3]. However, legume silages such as WPSs have low water-soluble carbohydrate (WSC) content, insufficient epiphytic LAB count, and high buffer capacity [
4]. These conditions favor undesirable fermentation (clostridial and enterobacterial) and reduce silage quality [
5].
Several studies evaluated microbial inoculant effects on WPS ensiling [
6,
7]. However, the delayed effect of microbial inoculant could not be sufficient to improve legume silage fermentation. Therefore, acid addition during the legume ensiling could be a promising strategy in ensiling for these materials [
8]. Formic acid is a well-known ensiling additive for low fermentable materials [
9]. Sodium propionate has been successfully utilized as a silage additive with a 0.35 benzoate equivalent [
10]. In addition, glycerol can be used as an LAB substrate [
11]. Wei et al. [
12] reported positive effects of formic acid and lactic acid inoculation in different crop silages. However, to the best of our knowledge, there is no study evaluating blends containing formic acid, sodium propionate, glycerol propionate, and glycerol with LAB inoculation instead of WPS ensiling. Therefore, we hypothesized that chemical and microbial additives could reduce fermentation losses, and chemical additives could improve nutritive value of WPS silage compared to LAB inoculation. The present study aimed to evaluate the effects of two chemical additives or a microbial inoculant on WPS silage fermentative profiles, microorganism counts, fermentation losses, chemical compositions, and protein degradability.
3. Results
Silage pH and NH
3-N concentration were lower (
p ≤ 0.021) and concentrations of lactic acid, acetic acid, butyric acid, valeric acid, isovaleric acid, and branched-chain fatty acids (BCFAs) were greater (
p ≤ 0.031) in ensiled material with additives than CON (
Table 2). Silage pH and concentrations of NH
3-N, ethanol, acetic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, and BCFA were greater (
p ≤ 0.016) in ensiled material with INO than with chemical additives (FA type and PA type). Comparisons between chemical additives revealed lower (
p ≤ 0.012) pH and concentrations of propionic acid, isobutyric acid, isovaleric acid, and BCFA in ensiled material with PA type than FA type. Counts of LAB, anaerobic bacteria, and mold and yeast were lower (
p < 0.001), and count of aerobic bacteria was higher (
p < 0.001), in silages with additives than CON. Counts of LAB and anaerobic bacteria were higher (
p < 0.001) and counts of mold and yeast were lower (
p < 0.001) in silages with INO than with chemical additives. Comparisons between chemical additives revealed greater (
p < 0.001) counts of LAB and anaerobic bacteria and lower (
p < 0.001) counts of yeast and mold in silage with PA in comparison to those with FA.
Gas losses (% DM) and total losses decreased (
p < 0.001), and DM recovery increased (
p < 0.001) when additives were incorporated to the silage, especially FA type and PA type (
Table 3). When comparing the type of silage additive (INO vs. chemical additives), gas losses, effluent losses, and total losses were lower (
p ≤ 0.039), and DM recovery was greater (
p < 0.001) for silages treated with chemical additives in comparison with INO. Gas losses were greater (
p = 0.032) for PA type than FA type. On the other hand, effluent losses were lower (
p ≤ 0.002) for PA-type silages in comparison with FA type. Dry matter recovery was greater (
p = 0.032) for FA-type than PA-type silages.
In general, incorporating additives to silages increased concentrations of DM, OM, CP, NFC, TDN, and NE
L and decreased concentrations of NDF in comparison to CON (
Table 4). Silage concentrations of DM, OM, CP, NDF, ADF, TDN, and NE
L increased (
p ≤ 0.019) by treating silages with chemical additives in comparison to INO. When the effects of chemical additives were contrasted, silages with PA type exhibited greater (
p ≤ 0.004) concentrations of NDF and lower concentrations of ADF, lignin, and NFC than silages with FA type.
Greater (
p = 0.006) proportions of A-fraction and lower (
p = 0.002) proportions of C-fraction were observed in silages treated with additives than CON (
Table 5). The effective degradability of silages was greater (
p < 0.001) in those treated with additives regardless of the passage rate used for calculations. Proportions of A-fraction and degradation rate of B-fraction were greater (
p ≤ 0.034), and proportions of B- and C-fractions were lower (
p < 0.001) in silages treated with chemical additives in comparison with INO. Effective degradability was greater (
p < 0.001) in silages treated with chemical additives in comparison with INO, regardless of passage rate used in calculations. Degradation rate of B-fraction was greater (
p < 0.001) for silages treated with PA type than FA type. Effective degradability of DM using passage rates of 50 and 80 g/kg/h in calculations was greater (
p ≤ 0.012) for silages with FA type than PA type.
When the effects of silage additives on protein fractions and effective degradability in each storage length were analyzed, silage A-fraction proportion was greater (
p ≤ 0.020) in silos with additives (INO, FA type, and PA type) in comparison with CON after 120 and 150 d of storage (
Table 6). Silage A-fraction proportion was greater (
p ≤ 0.041) for silages with chemical additives than INO after 60, 90, 150, and 180 d of storage. B-fraction proportion was lower (
p ≤ 0.028) in silages with chemical additives than INO after 90 and 150 d of storage. C-fraction proportions were lower (
p ≤ 0.020) in silages with additives in comparison with CON after 120 and 150 d of storage. After 120 and 150 d of storage, proportion of C-fraction in silages was lower when additives were applied. C-fraction proportions were greater (
p ≤ 0.019) in silages treated with INO in comparison with those treated with chemical additives after 60, 150, and 180 d of storage. The degradation rate of B-fraction in silages treated with chemical additives was greater (
p = 0.023) than in those treated with INO. Effective degradability of DM was greater (
p ≤ 0.004) when additives were applied in the silages in all storage lengths, except for 30 d. In general, effective degradability of DM was greater (
p ≤ 0.011) in silages treated with chemical additives in comparison with INO in all storage lengths, except for 30 d storage length and using a passage rate of 20 g/kg/h in estimations. Effective degradability of DM, regardless of feed passage rate, was greater (
p ≤ 0.019) in silages treated with chemical additives in comparison with those treated with INO only after 120 d of storage.
4. Discussion
It was hypothesized that silage additives would reduce DM losses and improve chemical composition and fermentation metabolites in WPS silage, whereas chemical additives would be more effective than INO in improving silage traits due to direct effect in decreasing pH. Indeed, chemical additives reduced silage DM losses, and additives improved chemical composition (i.e., greater CP and lower NDF concentration), increased lactic acid concentration, and reduced C-fraction proportion (undigestible CP) in WPS silage.
To the best of our knowledge, there is no study that compared fermentation traits of WPS ensiled with INO or chemical additives containing formic acid or propionic acid. In whole-plant corn silage, WSC concentration and pH at opening were similar between silage treated with LAB and with organic acids [
27]. In a mixed silage (alfalfa and perennial ryegrass), the pH was lower and WSC was greater in silages treated with formic acid regardless of the storage period (7, 15, or 45 d) in comparison with those treated with LAB [
28]. Regarding comparisons between chemical additives, silages treated with FA type had greater concentrations of NFC than those silages treated with PA type, aligning with lower silage pH at opening for FA silos in comparison with PA type. Formic acid is a stronger acid than propionic acid because of the inductive effect. Longer hydrocarbon chains exert a greater electron-pushing effect toward the carboxylic group, making the release of proton more difficult. Consequently, the easier the release of a proton, the stronger the acid [
29].
Chemical additives reduced NH
3-N content in WPS silage indicating lower proteolysis during silage fermentation, which is further confirmed by the greater concentration of CP observed in these silages when compared to other treatments. Aligning with the current study, authors have reported that chemical additives with organic acids are able to decrease ammonia N content in mixed legume–grass silage [
28]. It is important to note that fermentation parameters and metabolites were assessed at the opening of the silos, and they cannot represent the fermentation dynamics throughout the silage storage period. In general, additives increased lactic acid and butyric acid concentration and decreased acetic acid concentration in silage. These results contrast with counts of LAB, which were lowered by FA-type and PA-type treatments. It was anticipated that silages treated with INO would exhibit greater lactic acid content as the additive was composed of LAB. An inoculation rate of 10
5 to 10
6 microorganisms per gram of fresh forage is adequate for inoculated LAB to outcompete the epiphytic flora and establish themselves as the predominant population in silage [
30]. Organic acids present in chemical additives, however, should be able to rapidly decrease silage pH, thereby inhibiting bacteria activity and minimizing the production of fermentation metabolites, including lactic and other acids. Aligning with the later statement, lactic acid and anaerobic bacteria counts were decreased in silages treated with FA type. Gheller et al. [
31] observed decreased contents of lactic acid and acetic acid in whole-plant corn silage treated with chemical additives containing organic acids. The reasons for the increased silage content of lactic acid observed in the current study are not clear. Mold and yeast counts were decreased by adding silage additives, particularly due to INO and PA type effects. Microbial inoculants combined homofermentative LAB with
P. acidipropionici to minimize the aerobic spoilage of silages [
32]. Propionic acid bacteria metabolize sugars and lactic acid into acetic and propionic acids, compounds known to inhibit the growth of yeast and molds [
33]. The effect of INO in reducing counts of mold and yeast of WPS silage was previously reported [
34].
During the fermentation phases of silage, DM losses are derived from effluent and gas losses. Before the active fermentation phase, the oxygen trapped in the silo allows biological and chemical processes producing effluent, carbon dioxide, heat, and free NH
3-N [
35]. When silo becomes anaerobic, the losses are primarily from carbon dioxide production. The amount of DM loss depends on the dominant microbial species and the substrate fermented [
36]. Despite a significant effect on gas losses observed when contrasting CON with all silage additives, only chemical additives reduced losses and increased DM recovery. In addition, PA-type treatment was more effective in reducing effluent losses than FA. It is well known that chemical additives promote a rapid drop in silage pH with concomitant bacteriostatic and fermentation inhibitor effects, thus decreasing nutrient losses of the ensiled herbage [
3,
37]. The reasons for lower effluent losses in silages treated with PA type in comparison with FA type might be related to its ability in altering the cell permeability of microorganisms and competing with amino acids for space on active enzyme sites [
30]. Furthermore, silages treated with PA type exhibited lower counts of mold and yeast than FA-type-treated silages, which might have reduced aerobic deterioration. The literature lacks data on the effects of chemical additives with organic acids on WPS silage fermentation, but studies have reported improved DM recovery when different additives were included in whole-plant corn silage or in snaplage [
31,
38]. Agreeing with the current study, authors reported no effect of INO on WPS silage total losses [
34]. However, improved DM recovery or decreased total losses when the same microbial inoculant was applied in WPS silage was reported by Morais et al. [
7]. Inconsistent results can be related to phenological stage, which soybean plant was harvested (i.e., R6 vs. R7), and DM content of ensiled material (25.0 vs. 36.7% DM) when comparing the results of Morais et al. [
7] with those observed in the current study.
Regarding chemical composition, silages with chemical additives exhibited greater concentrations of DM, CP, and NFC, leading to increased NFC and TDN proportions. Silages with additives had lower concentrations of NDF, which is likely associated with lower solubilization or degradation of non-fiber components in comparison with CON. These results, especially differences in DM content, are supported by greater DM recovery of silages treated with chemical additives. Agreeing with the current study, authors reported greater concentrations of OM, CP, and NFC when ensiling whole-plant corn with different organic acid preparations composed mostly of formic acid and propionic acid [
31]. Other studies have also shown positive effects on nutritive value of silages treated with chemical additives with organic acids [
39,
40], even when compared to silages treated with microbial inoculant [
41]. Whole-soybean silages treated with INO, however, had lower concentrations of DM, CP, NFC, and TDN in comparison with silages with organic acids. Collectively, these results indicate that chemical additives are more effective in preserving chemical composition of the whole soybean plant. Although we did not measure the decrease in silage pH during the different phases of silage fermentation, it is somewhat expected that chemical additives promoted a rapid drop in silage pH in comparison with INO and thus interrupted the consumption of cellular content of plant cells by microbes faster than INO. In addition, silage pH at silos’ openings was lower in silages treated with organic acids than INO.
Concerning protein fractions in WPS silage, chemical additives (FA type and PA type) increased contents of A-fraction and decreased contents of B- and C-fractions, indicating low proteolytic activity in silos. Furthermore, chemical additives also increased digestion rate of B-fraction and effective degradability of CP. It is expected that increasing contents of A-fraction, which is promptly soluble in the rumen, and the digestion rate of the potentially degradable fraction (B-fraction) would increase effective degradability of silage. De Morais et al. [
7] reported greater proportions of A-fraction (572 g/kg) and lower C-fraction (45.3 g/kg) in WPS silage (R7 phenological stage) either in control group or treated with microbial inoculants. Contrasting with the current study, de Morais et al. [
7] observed a decrease in C-fraction proportion when microbial inoculants were incorporated into the silage stored for 120 d, which was associated with reduced fermentation losses and dilution of low degradable protein fraction. Differences in protein fractions between studies may be related to soybean cultivar, phenological stage at which plants were harvested, and crop conditions. In the current study, the lowest DM losses were observed in silages treated with chemical additives, which supports the decrease in silage C-fraction proportion with FA-type and PA-type additives.
Additives, especially the chemical additives, increased A-fraction and thereby de-creased B-fraction proportion (dilution effect) between 120 and 150 d of silage storage length. Thereby, in general, chemical additives increase protein-effective degradation. Controversially, Junges et al. [
42] reported that less than 5% of silage proteolysis is directly associated with acid production, and lower proteolysis has been well documented in lower-pH silages [
43,
44]. We are unaware of studies that evaluated the effects of chemical additives with organic acids on WPS silage protein fractions and solubility. Besides reduced NH3-N observed in acid-treated silos, structural changes induced by acid addition reduced estimates of effective degradation in the current study.