Effects of Ensiling Density on the Fermentation Profile and Aerobic Stability of Wilted Alfalfa Silage

: Silage quality and aerobic stability are the key factors affecting the utilization efficiency of silage feed, and ensiling density stands as the fundamental principle of silage making. The experiment presented here evaluates the effects of ensiling density on the silage quality and aerobic stability of alfalfa silage. In this experiment, alfalfa was harvested, wilted, chopped, and subsequently packed into 10 L laboratory silos. The ensiling densities were set to 800 g/L, 700 g/L, and 600 g/L, respectively, with three replicates in each group. Sampling and analysis were carried out at 45 days of silage and 8 days of aerobic exposure. The results showed that ensiling density significantly ( p < 0.05) affected the content of ammonia nitrogen, Flieg score, the counts of yeast, and mold. After 45 days of ensiling, 800 g/L silage had the highest contents of dry matter, water-soluble carbohydrates, crude protein, lactic acid, and total organic acids, and the lowest pH and ammonia nitrogen compared to the 700 g/L and 600 g/L silage ( p < 0.05). The Flieg score of 800 g/L silage was higher than those of the 700 g/L and 600 g/L silage. The counts of yeast and mold of 800 g/L silage was significantly lower than those of the 700 g/L and 600 g/L silage. During aerobic exposure, a consistent decrease in lactic acid and an increase in pH were observed among all silages. The aerobic stability of 800 g/L silage (156 h) was significantly higher than that in the 700 g/L (136 h) and 600 g/L silage (111 h). It was suggested that the increasing ensiling density above 800 g/L was an effective method to improve both the silage quality and the aerobic stability of alfalfa silage.


Introduction
Ensiling is an effective method of preserving nutrients by maximizing lactic acid production under anaerobic conditions.During ensiling, the ensiled material undergoes four aerobic stages: the field stage, the initial aerobic stage during ensiling process, the air-infiltration stage, and the aerobic deterioration stage after opening [1].In the early stages of ensiling, oxygen still exists within the silo, which stimulates the activity of aerobic microorganisms such as yeasts and molds, while also stimulating plant respiration (continuing metabolism of plant cells), causing a loss of both fermentation substrates and nutrients [2].In addition, inadequate compaction increases the volume of air trapped in silos and promotes the growth of undesirable bacteria.These undesirable bacteria vie with lactic acid bacteria for water-soluble carbohydrates and degrading proteins, causing an unpleasant odor and reduced palatability [3].Moreover, feeding spoiled silage to ruminants would cause reductions in intake and affect the health of livestock [4].During ensiling and feed-out, air also permeates in the peripheral areas of a silo; this could promote the growth of aerobic bacteria results in decreased aerobic stability [5].The high ensiling density could prevent air infiltration to silage mass and improve aerobic stability during aerobic exposure [6].Ruppel [7] found that, as packing density increased from 160 to 320 kg•m 3 , the dry matter losses decreased from 20% to 10%.Therefore, we can infer that packing density is negatively correlated with storage loss.
Alfalfa (Medicago sativa L.) is a high-quality leguminous forage in China, with high crude protein, vitamins, and a variety of minerals.It also has good palatability and high yield, so it is widely cultivated in many countries, and is considered an important diet for ruminants [3].Alfalfa is primarily used for producing silages to be fed to livestock [8].However, achieving high-quality silage in practice poses challenges due to its low dry matter and water-soluble carbohydrate contents, as well as its high buffering capacity [9].Therefore, proper wilt and adequate compaction are crucial measures for the production of high-quality alfalfa silage; these measures could reduce the moisture content of alfalfa and the amount of oxygen in the silos, thus inhibiting the growth of undesirable bacteria.
Ensiling density refers to the compactness or how tightly packed the forage is within a silo or bunker during ensiling [1].It is a critical factor that significantly influences the quality and preservation of silage.Ensiling density is determined by several key factors, such as the moisture content and the physical structure of ensilage materials [10].Diverse ensiling densities result in variations in the fermentation process and the quality of fermentation products [11].Ensiling density is often overlooked in practice, and the operation is not standardized, leading to poor-quality silage.Therefore, achieving the right ensiling density is crucial for fermentation efficiency and optimal nutrient retention [12].
Based on previous research, the compaction density of alfalfa silage in mass production should be 200-250 kg DM/m 3 , or there should be a clearance rate of less than 40%.This work proposes that higher ensiling densities lead to better quality and a longer aerobic stability time.Therefore, the purpose of this work to determine a suitable ensiling density for alfalfa silage, provide a reference for those engaged in production practices.

Preparation of Alfalfa Silage
The alfalfa was cultivated in Hemudu Town, Yuyao City (29.95 • N, 121.35 • E, 4 m asl, Zhejiang, China).The alfalfa was harvested (early bloom stage 5 November 2022) and wilted in the field (control the moisture content about 65%) and then carried to laboratory.The characteristics of wilted alfalfa are shown in Table 1.After chopping (2-3 cm) and mixing, the materials were loaded into 1 L and 10 L laboratory silos at different densities.The ensiling densities were set as 800 g/L, 700 g/L, and 600 g/L.Each density was replicated five times and stored at room temperature (16-21 • C).After 45 days of ensiling, the 1 L silos were opened to analyze the fermentation parameters and the aerobic stability of the 10 L silos was analyzed.

Chemical Component and Microbial Population Analysis
The silage sample was divided into two parts.Part one was placed in the oven at 65 • C for 48 h to dry to a constant weight; then, it was ground through a flour mill and passed through 40 mm and 100 mm screens.The dry matter (DM) was determined by Liu [6].Neutral detergent fiber (NDF), acid detergent fiber (ADF), acid detergent lignin (ADL) contents were determined by an ANKOM (A2000i) fiber analyzer.Water-soluble carbohydrate (WSC) content was determined by Zhao [13].
A measure of 200 g of silage was mixed with 600 mL of distilled water and left to soak at 4 • C for 24 h.Afterward, the mixture was filtered through two layers of gauze and a layer of filter paper.The resulting extract was then utilized to measure pH using a pH meter.Determination of lactic acid (LA), acetic acid (AA), propionic acid (PA), butyric acid (BA), and ethanol contents was conducted by an HPLC system (Agilent 1260 Infinity, Agilent Technologies, Inc., Waldbronn, Germany).The content of ammonia nitrogen (NH 3 -N) was determined by phenol-hypochlorite colorimetry [13].
Flieg's point was calculated by Abo-Donia [14].The formula is as follows: The lactic acid bacteria (LAB), aerobic bacteria (AB), mold, and yeast were counted using plate counting [15].All microbial data were transformed to log 10 for presentation and statistical analysis.

Aerobic Stability Test
After 45 days of ensiling, 45 silos were opened, and air was allowed to enter.Two layers of gauze were used to cover the tops of the silos, and the six temperature sensors of a data logger were placed in different geometric positions of the silos to record the temperature at 30 min intervals [16].Six probes were placed in the environment as blanks.The aerobic stability of silage was assessed by comparing the time required for the silage to exceed room temperature by 2 • C, and the fermentation parameters were determined during aerobic exposure at 2, 4, and 6 days.

Statistical Analysis
Statistical analysis was conducted using SPSS (version 13.0 for Windows; SPSS Inc., Chicago, IL, USA).Fermentation characteristics and microbial counts were analyzed via one-way analysis of variance (ANOVA) using the following model: In this model, Y i represents the dependent variable, µ denotes the overall mean, T i represents the effect of ensiling density, and e ij signifies the residual error term.
A factorial treatment design model was employed to analyze aerobic stability parameters via two-way analysis of variance, outlined as follows: where Y ij represents the dependent variable, µ denotes the overall mean, T i signifies the impact of ensiling density, D j indicates the effect of exposure days, (T × D) ij represents the interaction effect between ensiling densities and exposure days, and e ij is the residual error term.

Fermentation Quality of Alfalfa
Table 2 displays the fermentation parameters, microbial composition, and chemical compositions of alfalfa silage following a 45-day ensiling period.Ensiling density significantly affected (p < 0.05) the NH 3 -N content, Flieg score, and molds count.After 45 days of ensiling, the WSC content of 800 g/L silage (23.4 g/kg DM) was higher than that of the 700 g/L silage (20.1 g/kg DM) and the 600 g/L (12.9 g/kg DM) silage; the NH 3 -N contents of the 800 g/L and 700 g/L silages were significantly (p < 0.05) lower than that of the 600 g/L silage; the CP contents of the 800 g/L and 700 g/L silages were significantly (p < 0.05) higher than that of the 600 g/L silage.Ensiling density had little effect on the contents of DM, NDF, ADF, and PA.The 800 g/L silage had the lowest pH value (4.30), BA content (3.01 g/kg DM), and ethanol content (9.45 g/kg DM), and had the highest LA content (78.5 g/kg DM) and AA content (21.0 g/kg DM).The LA/AA values of the 800 g/L, 700 g/L, and 600 g/L silages were 3.63, 3.22, and 3.26, respectively.The Flieg scores of the 800 g/L and 700 g/L silages were 77.2 and 66.6, which are higher than the Flieg score of the 600 g/L silage (50.8).The LAB count of the 800 g/L silage (16.4 g/kg FW) was higher than those of the 700 g/L (13.8 g/kg FW) and 600 g/L (11.6 g/kg FW) silages.The yeast count of the 800 g/L silage (0.98 g/kg FW) was significantly (p < 0.05) lower than those of the 700 g/L (2.49 g/kg FW) and 600 g/L (3.84 g/kg FW) silages.The mold count of the 800 g/L silage was below the detected level.

Aerobic Stability of Alfalfa Silage
The alterations in fermentation parameters during exposure to aerobic conditions are depicted in Table 3.After 4 days of aerobic exposure, there were no significant (p > 0.05) changes in the pH of 800 g/L and 700 g/L silages.However, the pH of the 600 g/L silage increased sharply (p < 0.05) from 4.49 to 6.82.In all silages, the content of LA and AA gradually decreased; the NH3-N increased during aerobic exposure.On day 6 of aerobic exposure, the following changes were found: the LA contents of the 800 g/L, 700 g/L, and 600 g/L silages decreased by 57.5, 51.6, and 39.2 g/kg DM; the AA contents decreased by 11.4, 16.1, and 20.7 g/kg DM; the NH3-N contents increased by 129, 135, and 153 g/kg TN.The content of PA and BA increased slowly in all silages during aerobic exposure.The changes in microbic counts during aerobic exposure are shown in Table 4.The LAB count had a decreasing trend and the AB and yeast counts increased continually in all silages.The 800 g/L silage had the highest LAB count (10.7 log 10 cfu/g FW) and the lowest AB and yeast counts (10.1 and 6.32 log 10 cfu/g FW) during aerobic exposure.Data were means of five replicates.a-c means in the same row followed by different letters are significantly different (p < 0.05).FM, fresh matter; LAB, Lactic acid bacteria; AB, Acetic acid bacteria.SEM, standard error of means; T, treatments; D, aerobic exposure days; T × D, interaction between treatments and aerobic exposure days.
Figure 1 illustrates the aerobic stability of alfalfa silages.The silage temperatures of the 800 g/L, 700 g/L, and 600 g/L silages were different by more than 2 • C from the ambient temperature after 156, 146, and 111 h during aerobic exposure, respectively.

Dimension Reduction Analyses
Principal component analysis (PCA) was employed to enhance the visualization of the correlation among chemical compositions, microbial counts, and aerobic stability (Figure 2).Principal component 1 (PC1) and principal component 2 (PC2) accounted for 42.6% and 23.4% (totaling 66%) of the total variance, demonstrating that these two principal components effectively captured the sample information.The high density had a higher contribution to the water-soluble carbohydrates content, LAB count, and aerobic stability.

Dimension Reduction Analyses
Principal component analysis (PCA) was employed to enhance the visualization of the correlation among chemical compositions, microbial counts, and aerobic stability (Figure 2).Principal component 1 (PC1) and principal component 2 (PC2) accounted for 42.6% and 23.4% (totaling 66%) of the total variance, demonstrating that these two principal components effectively captured the sample information.The high density had a higher contribution to the water-soluble carbohydrates content, LAB count, and aerobic stability.

Dimension Reduction Analyses
Principal component analysis (PCA) was employed to enhance the v the correlation among chemical compositions, microbial counts, and ae (Figure 2).Principal component 1 (PC1) and principal component 2 (PC2) 42.6% and 23.4% (totaling 66%) of the total variance, demonstrating t principal components effectively captured the sample information.The hig a higher contribution to the water-soluble carbohydrates content, LAB coun stability.

Discussion
During the early stage of ensiling, the DM and WSC contents are influenced by the microbial activities and respiration of plant cells [1].Thus, the higher-density silage had a lower DM loss [7].The 800 g/L silage had the highest WSC and DM contents; this is due to the fact that high density could reduce the volume of air in the silo, which reduced the consumption of WSC and other nutrients by plant cell respiration and aerobic bacteria [17].NH 3 -N production is indicative of the degree of protein degradation within silage; this reflects the extent of proteolysis and the fact that the NH 3 -N content of well-preserved silages should be lower than 80 g/kg TN [1].The results show that the 800 g/L silage had good conservation, and the high-density silage had the highest CP and the lowest NH 3 -N contents.This indicated that the high density inhibited the growth of undesirable bacteria and thus reduced the degradation of proteins [18].Compared with raw materials, there were differences in CP, NDF, and ADF contents among all silages at 45 days of ensiling; this was probably because the different ensiling conditions stimulated the release of plant enzymes and promoted the degradation of cellulose and hemicellulose [19].In addition, the ensiling process usually produces a certain temperature, which is conductive to the growth and activity of microorganisms; higher temperatures may promote protein breakdown and volatilization, thus reducing the crude protein content in plant materials [1].
The epiphytic LAB values present on fresh alfalfa exceed the minimum threshold required for fermentation (10 5 cfu/g FW) [20].The high compaction could promote plant cell rupture and juice exudation and might promote LAB fermentation, quickly producing more LA and leading to a decreased pH [21].It was found that the 800 g/L silage had a higher LA content and a lower pH than the 700 g/L and 600 g/L silages [22].The LA/AA values of all silages were found to be above 3; this indicates that the fermentation process was predominantly homolactic [1], resulting in reduced fermentation losses, particularly in silages with high ensiling densities [23].The research showed the ensiling density could not change the type of fermentation of the silage; silages with different ensiling densities were found to have similar bacterial communities [19].However, the 800 g/L silage had the highest count of LAB and the lowest count of yeast and mold.This finding indicated that high ensiling density rapidly created an anaerobic environment for the growth of LAB.Meanwhile, the highly acidified environment inhibited the growth of aerobic bacteria and yeast [24].Kung [23] showed that a lower packing density slowed down the rate of fermentation and acidification; the abundance of yeast was found to increase during ensiling.The Flieg score, derived from the dry matter content and the pH observed in the silages, serves as a method for evaluating the fermentation quality of silage.These factors do not take into account organic acids, the extent of protein degradation, or the aerobic stability of the silage [25].In the work, the Flieg scores of all silages were higher than 61, indicating that all silages had been well preserved [14].
During the process of opening and feeding silage, air exposure is unavoidable.Aerobic microorganisms such as yeast and mold can proliferate rapidly.These aerobic microorganisms metabolize carbohydrates and organic acids to produce carbon dioxide and water, generating heat and causing a rapid increase in pH [16].Therefore, changes in pH and temperature are used as crucial indicators of aerobic deterioration.In the experiment, the pH, the contents of NH 3 -N and PA, and the counts of AB and yeast increased; meanwhile, the contents of LA and AA and the LAB count decreased in all silages during air exposure.These results showed that all silages experienced varying degrees of aerobic deterioration [26].The 600 g/L silage exhibited elevated levels of LAB, AA, and yeast compared to the other silages.Among these microorganisms, AA and yeast are primarily responsible for initiating aerobic deterioration in silage, leading to nutrient degradation and the release of noxious odors and harmful substances.Additionally, during aerobic spoilage, mold-produced mycotoxins pose risks to livestock health, potentially impairing liver function and weakening the immune system.Furthermore, through the food chain, these mycotoxins can accumulate and threaten human food safety.
The 800 g/L, 700 g/L, and 600 g/L silages surpassed the ambient temperature by 2 • C after 156, 136, and 111 h, respectively; these results indicated that tightly packed silage was more stable than loosely packed silage [27].Exposure of silages to air triggers heightened aerobic microbial activity, with lactic-acid-assimilating yeasts initiating the process of aerobic deterioration.This phenomenon culminates in the generation of excessive heat [28].This corresponds to the results which showed that lactic acid content decreased, and the temperature of the silage mass increased during aerobic exposure.The 800 g/L silage exhibited significantly greater aerobic stability compared to the 700 g/L and 600 g/L silages.This illustrates that high ensiling density correlates with a longer duration of aerobic stability compared with the low ensiling density.This is primarily due to the lower porosity and permeability of high ensiling density, which makes it difficult for oxygen to penetrate in the interior of the silage [29].
In this work, 800 g/L is the maximum capacity of the laboratory silo; the silo is quickly filled, densely packed, and sealed immediately during ensiling to minimize the exposure of forage and silage to air.However, it is difficult to completely exclude air from the silage mass during silo filling and prevent its infiltration during storage in farm-scale silos.Therefore, producers have to know the capacity of their silos to determine the maximum amount of forage that the silos should be filled with.In addition, producers need to pay attention to the degree of crushing for forage; pre-processing treatments such as shredding appear advantageous for the production of high-quality silages [30].Generally, producers follow the standard operation method to ensure the production of high-quality silage.

Conclusions
A high ensiling density effectively enhances the fermentation quality and aerobic stability of silages; this was evidenced by higher dry matter and lactic acid content, along with lower pH and NH 3 -N levels.The Flieg score of all alfalfa silages was greater than 60 points, indicating that all silages underwent satisfactory fermentation.In conclusion, the experiment confirms the previous hypothesis, and it was suggested that increasing the ensiling density above 800 g/L was an effective approach for enhancing both the fermentation quality and the aerobic stability of alfalfa silage.

Figure 1
Figure1illustrates the aerobic stability of alfalfa silages.The silage temperatures of the 800 g/L, 700 g/L, and 600 g/L silages were different by more than 2 °C from the ambient temperature after 156, 146, and 111 h during aerobic exposure, respectively.

Figure 1 .
Figure 1.Temperature changes (a) and aerobic stability (b) of alfalfa silages after exposure to air.ab means different letters are significantly different (p < 0.05).

Figure 2 .
Figure 2. Principal coordinate analysis (PCA) plots for chemical compositions, microbial counts, and aerobic stability of alfalfa silage after 45 days.PC1 explains 42.6% of the variance; PC2 explains 23.4% of the variance.Black triangles are high-density silage; red triangles are low-density silage; green triangles are medium-density silage.

Figure 1 .
Figure 1.Temperature changes (a) and aerobic stability (b) of alfalfa silages after exposure to air.ab means different letters are significantly different (p < 0.05).

Figure 1 .
Figure 1.Temperature changes (a) and aerobic stability (b) of alfalfa silages after ex ab means different letters are significantly different (p < 0.05).

Figure 2 .
Figure 2. Principal coordinate analysis (PCA) plots for chemical compositions, micro aerobic stability of alfalfa silage after 45 days.PC1 explains 42.6% of the varianc 23.4% of the variance.Black triangles are high-density silage; red triangles are low green triangles are medium-density silage.

Figure 2 .
Figure 2. Principal coordinate analysis (PCA) plots for chemical compositions, microbial counts, and aerobic stability of alfalfa silage after 45 days.PC1 explains 42.6% of the variance; PC2 explains 23.4% of the variance.Black triangles are high-density silage; red triangles are low-density silage; green triangles are medium-density silage.

Table 2 .
Fermentation profile and microbial and chemical compositions of alfalfa silage after 45 days of ensiling.
Data were means of five replicates.a,b means in the same row followed by different letters are significantly different (p < 0.05).DM, dry matter; FW, fresh weight; TN, total nitrogen; log 10 : decimal logarithm; cfu, colony-forming units.SEM, standard error of means.

Table 3 .
Changes in pH, NH3-N, organic acid, and ethanol of alfalfa silage during air exposure.
Data were means of five replicates.a-c means in the same row followed by different letters are significantly different (p < 0.05).DM, dry matter; TN, total nitrogen; SEM, standard error of means; T, treatments; D, aerobic exposure days; T × D, interaction between treatments and aerobic exposure days.

Table 4 .
Changes in the microbial counts of alfalfa silage during air exposure.