4.2. Crude Protein Fractions
Crude protein fractions in fresh lucerne material were in a typical range for this forage legume, although the present proportion of NPN compounds of 259 g kg
−1 CP was substantially higher than literature data, i.e., 150 g kg
−1 CP [
3], 170-183 g kg
−1 CP [
37] and 180–190 g kg
−1 CP [
35]. It may be noted that the highest discrepancy in NPN proportion, found between the results of the present study and of Guo et al. [
3], might partly also derive from different methodologies. In the present study, tungstic acid was used to precipitate TP, which cuts off peptides of an approximate chain length of more than three AA [
17]. Guo et al. [
3], however, used trichloroacetic acid to precipitate TP, which cuts off at about 10 AA [
17].
The pattern of CP fractions in LS with NPN being the largest, and moderate ruminally degradable TP being the second largest proportion of total CP corresponded to the literature [
3,
35]. It is notable that NPN contents of present LS were similar to those from Broderick [
2] and Seale et al. [
10], but higher than values reported by others, for instance, 684 g kg CP
−1 [
3] or 599 g kg CP
−1 [
38] in untreated LS. However, these NPN values were determined after only 35 or 30 days of ensiling, respectively, probably underestimating NPN in LS as intrinsic protease and carboxypeptidase were recently shown to remain largely active for more than 30 days after ensiling [
38]. Therefore, NPN values of the present LS, which were stored for 120 days, might provide a more realistic insight and should be considered when comparing different results or designing experiments for silage additive evaluation in LS. In this context, it may be noted that the Federal Working Group for Forage Preservation (Bundesarbeitskreis Futterkonservierung; [
12]) recommends at least 90 days of ensiling for any silage-related experiment, e.g., when evaluating the effect of silage additives. Besides, the influence of the cut number should also be taken into account as NPN was 10% higher in third-cut LS when compared to NPN proportion of first-cut LS from the same sward [
35]. Likewise, present LS was produced from a third cut, which thus may have been a contributing factor and should be investigated in future studies.
The SU reduced NPN along with increasing moderately ruminally degradable TP, which was likely caused by faster and stronger acidification, consequently suppressing proteolytic microorganisms in the silos [
11]. These observations were in accordance with Seale et al. [
10] who analyzed the effect of glucose and fructose addition with or without microbial inoculants on LS. However, in Italian ryegrass (
Lolium multiflorum LAM.), Heron et al. [
39] found that plant-derived proteases remained active over a wide pH range, which is also true for lucerne with major endopeptidases having optimum activities at pH 4 [
40]. Thus, despite SU treatment and probably rapid acidification, the relevant plant-derived proteolytic activity may have taken place, particularly in 250DML LS.
An effect of WI was found for NPN concentration, which was higher in LI LS. Likewise, high-intensity wilted grass silages had approximately 100 g kg
−1 CP lower NPN proportions compared to low intensively wilted grass silages [
9] and, together with the present observations, demonstrate the TP stabilizing effect of HI treatments. The rapid inhibition of plant-derived proteases, which depend on sufficient water availability [
5], may be causative. In this context, Owens et al. [
41] produced LS with a DM of 350 g kg
−1 and observed reduced NPN amounts of approximately 50 g kg
−1 total N in those LS that needed shorter wilting periods to reach the desired DM, which thus can be ascribed to a higher WI. Likewise, when wilting times were different due to varying levels of shade during wilting, they also observed an increase of NPN with shade, thus substantiating the TP stabilizing effect of an HI treatment. However, it must be considered that although there is evidence for a reduction of plant-derived protease activity by HI treatment, it is very arguable whether plant enzymes were completely deactivated as the moisture loss was only until a DM content of maximal 350 g kg
−1. Thus, plant-derived proteases may still have contributed to overall proteolytic processes resulting in the still substantial conversion of TP to NPN in high-intensity wilted LS.
The DML treatment also effected NPN proportion, which was lower in 350DML LS. This confirmed previous findings [
9,
42] and may be explained by a lower water activity in the silos, consequently reducing microbial metabolism [
34]. However, this mechanism should be even more pronounced at DM contents above 500 g kg
−1 [
9]. The effect of DML on slowly ruminally degradable TP may be of marginal importance as this fraction could not be determined in six of eight LS. The higher contents of moderately ruminally degradable TP in 350DML LS, however, may be beneficial regarding the quality of CP that is provided to the animal, meaning a decelerated ruminal CP degradation and therefore potentially improved N utilization by rumen microorganisms. As obtained for the TP proportion of 350HISU, the combination of HI, elevated DML and SU should have limited both plant-derived and microbial CP degradation and thus most effectively stabilized the TP content in the present study.
4.3. Amino Acids
A variety of factors influence the AA composition in silages, including wilting rate, acidification, and the microbial activity in the silo, but also plant-associated factors like tannin concentration or activity of plant proteases [
5]. To the authors’ knowledge, information about the effects of WI, DML, or SU on AA composition of LS is rare [
3,
43]. However, as the vast majority of AA is degraded in the rumen, knowledge on AA profiles seems to be more important for feedstuffs with high ruminally undegradable CP [
9], which does not apply to the present LS. Though, it is worthy of remark that pre-ensiling treatments clearly effected the AA composition of LS. For instance, the higher proportions of free His, Asp, Lys, Thr, Glu, and Pro in 350DML LS should be the result of reduced microbial activity [
5]. Likewise, a similar pattern was observed for total AA. The reducing effect of SU on total free AA content supported the TP preserving effect that was also observed for the distribution of CP fractions and should be caused by rapid acidification [
5].
Biogenic amines are predominantly formed during proteolysis in silages [
44] and Ohshima and McDonald [
45] described the decarboxylation of Glu to GABA during lucerne ensiling, which is reflected by the lower Glu concentrations in the present LS without SU. As summarized by Scherer et al. [
44], biogenic amines are associated with lower feed intake and potential impairments to animal health. Thereby, GABA is an important biogenic amine and known to act as a neurotransmitter. It is also involved in the sensation of pain and anxiety as well as neurological diseases [
46]. Although there is no clear trend for the effect of GABA on feed intake [
44], a negative correlation between feed intake and total amine concentration has been observed [
47] and the reduced GABA concentration in LS with 350DML or SU may, therefore, be interpreted as beneficial; particularly also because of potential health risks when biogenic amines would be absorbed by host animals, who, however, are more susceptible under acidotic conditions [
48].
4.5. Fermentation Pattern
The pH values were lower for SU LS, which was reflected by higher lactic acid concentration in these silages. Without the SU treatment, the high buffering capacity of lucerne [
7] may have hindered rapid and strong acidification and consequently resulted in higher pH. In this context, the higher ammonia-N contents should also be considered, which can limit the pH drop in silages, as well [
50]. Besides, low lactic acid concentration may be caused by metabolic activities of lactate-utilizing lactobacilli [
51]. Owens et al. [
7] stated a pH below 5.0 as a threshold to maintain forage quality and limit protein degradation in the silo, which thus was only met by SU LS. Likewise, the pH of these silages was within the common range for legume silages at this DM content [
50]. Water-soluble carbohydrates [
52] as well as total non-structural carbohydrates [
7] decrease during wilting of lucerne due to plant enzyme activity and respiration, which are both reduced by moisture loss [
53]. Therefore, there should have been less WSC degradation in the plant material undergoing HI treatment and consequently, a stronger pH drop along with increased lactic acid concentrations in HI LS was initially expected. However, the lack of a wilting effect may be explained by the overall very low WSC concentration of lucerne [
6], which further was cut in the morning when WSC concentrations are again lower compared to the afternoon [
7].
An impact of DM content on silage pH was often described in the literature [
50,
34]. Thereby, silages with DM contents below 300 g kg
−1 are extremely susceptible to clostridial fermentation [
50], which results in elevated pH values as well as high butyric acid concentrations. In the present study, however, DM content had neither an effect on silage pH nor on butyric acid concentration. Possibly the SU treatment superimposed a potential effect of DML, which is indicated by closer examination of butyric acid concentrations, which were numerically but not statistically significantly higher in 250DML LS. Moreover, the influence of DM content on clostridial fermentation and thus silage pH is more pronounced at DM contents of 400 g kg
−1 or more [
11], which is confirmed by the findings of Santos and Kung [
34].
The lower ammonia-N concentration in SU LS further strengthens the assumption that addition of rapidly fermentable carbohydrates better inhibited degradation of nitrogenous compounds in these LS compared to LS without SU, and similar trends have been observed previously [
10]. Regarding the impact of DML, ammonia-N concentration is generally higher in wet silages, which corresponds to present findings and is often ascribed to clostridial fermentation [
50]. Likewise, reduced ammonia-N contents in LS with high DM contents were also observed by Santos and Kung [
34]. Thus, a greater WI seemed to preserve CP from degradation in the silo. However, according to Wyss et al. [
35], ammonia-N proportions lower than 100 g kg
−1 N is preferable for LS. This threshold was not met in the present study, even for 35HISU. In comparison to fresh-cut lucerne [
35], plant material of the present study already showed a higher NPN proportion before ensiling, and may explain the high ammonia-N concentration in LS, irrespectively of applied pre-ensiling treatment. However, the increase of NPN from fresh-cut material to silage material were on the same level in the study by Wyss et al. [
35] and the present study. Regarding the high ammonia-N concentration in the present LS, the variation of CP composition between different lucerne cultivars [
54] should be considered, as well. Moreover, it can be speculated whether chopping of plant material subsequently supporting silo compression would have increased TP proportions as it was described earlier [
50,
55]. However, as LS was prepared according to recommended guidelines [
12], the latter point may be of minor importance. Besides, higher ammonia-N concentration is assumed to be associated with undesirable metabolites like biogenic amines [
50], which is in line with the present observations for higher GABA concentration in 250DML LS.
High concentration of acetic acid is associated with high DM and energy losses [
50] as well as considerably reduced ad libitum feed intakes [
56]. Compared to the literature [
34,
35], the acetic acid contents of LS in the present study can be classified as slightly high for 250DML LS and thus would negatively impact their nutritive value. Increased activity of Enterobacteriaceae [
57], as well as increased deamination [
5], could be causative for acetic acid formation, which is further favoured by high moisture contents [
50] and in accordance with the higher acetic acid concentration in 250DML LS. However, the presence of acetic acid is not a disadvantage per se. The average 32 g kg
−1 DM acetic acid in the 350DML LS, however, might be regarded beneficial as such concentrations have a positive effect on aerobic stability of silages [
58].
A butyric acid concentration higher than 5 g kg
−1 DM indicate elevated clostridial activity and due to high losses of energy, this means diminished energy supply to the animal and, consequently, performance may suffer [
50]. This threshold was not exceeded for 350DML LS and only applies to 250HI and 250LI. Likewise, these two treatments also had the highest ammonia-N concentration, which further points to clostridial fermentation [
50]. Together with the observation that 250HI and 250LI did also not meet the pH threshold for maintaining forage quality in the silo [
7], these two LS should, therefore, be classified as poor-quality silages and potentially spoiled material.
Concerning ethanol, SU tended to increase this alcohol in the silages. Though, ethanol concentration was low for all LS and thus does not indicate elevated yeast metabolism [
50,
59]. Minor amounts of ethanol can also originate from heterofermentative lactic acid fermentation [
60], which can never be fully prevented during ensiling. Weiß and Kalzendorf [
52] observed higher concentrations of ethanol and ester compounds in LS with low DM contents and further postulated a positive correlation between ethanol and ester concentration in silages, which is both confirmed by the present findings for ethanol and ethyl lactate as well as ethyl acetate. The effect of esters in silages is not fully clear [
47,
61], but negative correlations to short-term DM intake were observed earlier [
61]. Thus, despite lower NPN proportions in SU LS, the effect of SU on ester occurrence could be regarded as critical.
4.6. General Considerations
Up to now, WI has not received much attention in silage preparation and studies investigating the effect of different WI on silage characteristics are rare. It has been reported that wilting per se effects CP composition, for instance by reducing ammonia-N contents in LS [
54]. Thus, applying HI treatments may even be more effective for stabilizing TP content in LS, which is underlined by the present findings that confirm our hypothesis of a TP preserving effect by the HI treatment. A variety of silage additives exists that limit proteolysis in LS [
38]. However, they cause costs for acquisition, and in case of organic acids, also for maintenance of corroded machinery and concrete [
62]. In contrast, HI treatment does not require additional application systems or further technical equipment and in this regard is an easy to apply tool for improving the quality of on-farm produced protein, and consequently might help to reduce costs for ruminant diets and increase sustainability. Additionally, feed intake of wilted grass silage was increased when compared to non-wilted [
63]. In case this also applies to LS, a superior energy and nutrient provision to the animal may be achieved by intensively wilting lucerne to higher DML; provided that mechanical losses during harvest do not exceed the benefits of HI. Regarding the practicality of HI treatment, if possible high solar radiation along with high wind speed should be present during lucerne harvesting. However, this cannot always be guaranteed, which restricts the practicability of HI treatments. To support the effect of rapid dehydration, maceration can be an effective addition to further increase the wilting rate [
64] or to compensate weather conditions that may not be as ideal for HI as described in the present study. However, the risk of mechanical losses and thereby associated nutrient changes can be higher when using maceration [
64], which needs to be taken into account. Besides, artificial drying treatments are surely a more weather independent option to obtain similar TP preservation [
65] as here found for HI in the sun. However, increased production costs due to high energy demands may outweigh the beneficial effects [
36] of artificial drying on CP composition.
Concerning the effects of SU on fermentation and CP quality, provision of rapidly fermentable carbohydrates is recommended. Particularly because an inoculation with lactic acid producing bacteria alone may not improve the situation as long as there is not enough easily accessible substrate for lactic acid fermentation [
10]. Thus, relating to large scale on-farm conditions, mixing lucerne crop with molasses, crushed cereals, or high WSC forage species before ensiling may constitute a method for equivalently substituting SU treatment in LS. A delayed cutting of lucerne in order to increase non-structural carbohydrates, particularly starch, may not be appropriate as Owens et al. [
7] did not find a protein preserving effect in LS differing in WSC content due to different cutting times during the harvest. However, present results revealed concerns about promoted ester and ethanol formation in SU LS that should be kept in mind and require further investigation.
Finally, the pre-ensiling treatment combination of all three factors, i.e., HI treatment to high DML with SU, has the strongest potential to reduce the extent of CP degradation during ensiling, thus improving the protein value and potentially increasing ruminal N retention, particularly when combined with an appropriate carbohydrate source.