Journal of Animal Science and Technology
Korean Society of Animal Sciences and Technology

Effects of sodium diacetate and microbial inoculants on fermentation of forage rye

Yan Fen Li1, Eun Chan Jeong1, Li Li Wang1, Hak Jin Kim2, Farhad Ahmadi2, Jong Geun Kim1,2,*
1Graduate School of International Agricultural Technology, Seoul National University, Pyeongchang 25354, Korea
2Research Institute of Eco-Friendly Livestock Science, Institute of GreenBio Science Technology, Seoul National University, Pyeongchang 25354, Korea
*Corresponding author: Jong Geun Kim, Graduate School of International Agricultural Technology, Seoul National University, Pyeongchang 25354, Korea. Tel: +82-33-339-5728, E-mail:

© Copyright 2023 Korean Society of Animal Science and Technology. This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Received: Jun 12, 2022; Revised: Oct 13, 2022; Accepted: Oct 23, 2022

Published Online: Jan 31, 2023


Rye (Secale cereale L.) is a valuable annual forage crop in Korea but there is limited information about the impact of chemical and biological additives on fermentation characteristics of the crop. This experiment was conducted to investigate fermentation dynamics of wilted forage rye treated with the following six additives; control (no additive), sodium diacetate applied at 3 g/kg wilted forage weight (SDA3), 6 g/kg wilted forage weight (SDA6), inoculations (106 CFU/g wilted forage) of Lactobacillus plantarum (LP), L. buchneri (LB), or LP+LB. The ensiled rye sampled at 1, 2, 3, 5, 10, 20, 30, and 45 days indicated that the acidification occurred fast within five days of storage than the rest of the storage period. The microbial inoculants decline the pH of ensiled forage, more rapidly than the control or SDA treated, which accompanied by the decrease of water-soluble carbohydrates and increase of lactic acid. Compared with the control silage, all treatments suppressed ammonia-nitrogen formation below to 35 g/kg DM throughout the sampling period. Suppression of total microbial counting occurred in SDA6, LP, and LP + LB. The lactic acid production rates were generally higher in microbial inoculation treatments. Acetic acid concentration was lowest in the LP-treated silage and highest in the SDA- and LB-treated silages. The in vitro dry matter (DM) digestibility and total digestible nutrients were the highest in the silage treated with SDA (6 g/kg) at day 45 of ensiling. Based on lower ammonia-nitrogen concentrations and higher feed value, ensiling forage rye treated with SDA at 6 g/kg is promising through enhanced silage quality.

Keywords: Conservation; Microbial inoculant; Sodium diacetate; Wilting; Winter rye


Winter rye is an important cold- and drought-tolerant cereal crop that can remain productive in less fertile lands with a wide range of soil pH. Moreover, winter rye has several ecosystem benefits such as prevention of soil erosion and elevation of soil microorganism activities [14]. As a silage crop, winter rye can be harvested earlier to achieve higher quality forage (like early heading stage); early harvest also allows for another crop to be planted in a double-cropping system [5]. However, the high moisture concentration of forage rye at the vegetative stage is unsuitable for ensiling because of excessive silage effluent, soluble nutrient loss, and potential clostridial spoilage. Those factors sometimes affect the rye silage quality [6,7]. Field wilting has been recommended to optimize the conditions for silage fermentation [8]. Wilting reduces moisture concentration in forage, minimizing protease activity, and seepage from ensiled forage [6]. Recently, Zhao et al. [9] noted that the population of lactic acid bacteria (LAB) increased when forage rye cut at the heading stage was wilted for 24 h, resulting in more desirable fermentation patterns during ensiling.

Despite the high concentration of soluble sugars in small-grain forages, the likelihood of producing high-quality silage can be increased by using biological and chemical additives, particularly when the forage dry matter (DM) concentration at ensiling is suboptimal [5,10,11]. Moreover, the hollow stems of winter forage rye may provide air space during ensiling and create aerobic environment in silo [12]. As a result, the use of additives is recommended to promote rapid acidification, resulting in suppression of unwanted microorganism growth at the early ensiling stages.

Sodium diacetate (SDA) is an FDA-certified preservative capable of inhibiting microbial growth and, thus improving silage fermentation quality [13,14]. Organic acid-based additives, such as SDA, are readily ionized to acetic acid and immediately acidify forage biomass, thereby suppressing the growth and activity of unwanted organisms during the early phase of fermentation and minimizing the loss of nutrients during ensiling [6,15,16]. In their undissociated form, organic acid salts can readily permeate the cells of yeast and mold, and release hydrogen ions within the intracellular region. This increases adenosine triphosphate (ATP) expenditure of unwanted organisms for maintenance of intracellular homeostasis, and thus disrupts their cellular metabolism [1719].

Although the presence of epiphytic LAB on forage biomass surfaces can naturally initiate ensiling fermentation, microbial inoculants are usually recommended to support rapid acidification, which improves fermentative quality, and minimizes nutrient degradation during fermentation [20,21]. Previous studies identified that microbial inoculants such as Lactobacillus plantarum (LP) and L. buchneri (LB), individually or in combination, enhanced the fermentative quality of cereal grain and grass silages [2224]. However, inoculation with both homofermentative and heterofermentative LAB has provided inconsistent results when tested at variable moisture levels [25]. A meta-analysis also concluded that the improvement in fermentation quality was achieved by crop-specific application of homofermentative or heterofermentative LAB [26]. These results indicate the necessity of additional information to clarify the individual or combined effects of the LAB inoculants on the fermentation dynamics of wilted forage rye at the vegetative stage.

Information is limited comparing the usefulness of chemical- and biological-based additives for enhancing the silage fermentation quality of wilted forage rye. Therefore, this experiment was designed to determine the effects of microbial inoculants, including LP, LB, and their combination, and an organic acid preservative (SDA) on the dynamics of the anaerobic fermentation and nutrient conservation in wilted forage rye through monitoring fermentation process.


Forage rye production and wilting

Rye (Secale cereale L.) was seeded in October 2019 at the experimental field of Pyeongchang Campus, Seoul National University (37°32ʹ46.1ʺ N, 128°26ʹ17.9ʺ E) in Republic of Korea. The temperature and rainfall during the growing season are presented in Fig. 1. Forage rye was harvested at the early heading stage (May 13, 2020) with a hand clipper. Five randomly chosen spots (1 m × 1 m) were harvested to estimate forage production. The forage rye was harvested at 6-cm stubble height and wilted on the field for 24 h, tedded in 12-h interval.

Fig. 1. Average temperature and precipitation from October 2019 to May 2020). [65]
Download Original Figure

The wilted whole-plant rye was chopped into a theoretical cutting length of 20–30-mm using a forage cutter (Richi Machinery, Henan, China) and thoroughly mixed. The forage mass was divided into six equal portions, and randomly allocated to one of the following treatments: SDA3, SDA6, LP, LB, and LP + LB. The SDA (99%; Shanghai Rhawn Chemical Technology, Shanghai, China) was applied at rate of 3 (SDA3) and 6 g/kg wilted weight (SDA6). The microbial inoculants were LP (NLRI-101, LB (ATCC4005), and the combination (LP + LB; 1:1 ratio) of the two lactobacilli. The application rate was 1 × 106 colonial forming unit (CFU) per g wilted mass. A control treatment (without any additives) received 10 mL distilled water per kg wilted biomass. Other five additives were sprayed uniformly on forage mass with dispensing the same volume of distilled water as the control. After manually mixing, a 400-g mass of forage was packed in a plastic vacuum bag (Food grade, 28 cm × 36 cm, Korea), sealed by a vacuum packer (FM-06, Aostar, Seoul, Korea). All the silage bags were stored at 20°C–22°C. The 0-day ensiling was sampled immediately after the preparation of all treatment silages. The stored silage treatments were opened at 1, 2, 3, 5, 10, 20, 30, or 45 days of ensiling. The sampling days were set according to Santos et al. [27]. At each sampling day, the whole ensiled biomass in a bag was mixed thoroughly and split into three equal amounts for later analyses. The first portion was immediately frozen (–80°C) for later analysis of ammonia-nitrogen (NH3-N), lactic acid, and acetic acid. The second portion was kept fresh for immediate measurement of pH, LAB, mold, and total microorganisms. The third portion was weighed fresh, dried in an oven at 65°C for 72 h for determination of DM and feed values.

Analytical procedures

Silage extract was prepared to determine the fermentation profile of rye silage [28]. The acidity of the extract was determined immediately after opening the silo bags with an AB 150 pH meter (Fisher Scientific International, Hampton, NH, USA). Quantification of NH3-N was undertaken using a UVIDEC-610 spectrophotometer (Jasco, Tokyo, Japan) [29]. Lactic acid and acetic acid were analyzed by HPLC (Detector, RI; Column, Agilent Hi-Plex H; Agilent Technologies 1260 Infinity, Santa Clara, CA, USA), according to the procedure specified before [30]. LAB, mold, and total microorganisms were counted by streaking agar plate method [31] with a detection limit of 2 Log10 CFU/g wet weight, as described before [28].

The dried samples were ground into 1-mm particle size using a cutting mill (Thomas Scientific, New Jersey, USA) to determine nutrient value of rye silage. Neutral detergent fiber (NDF) and acid detergent fiber (ADF) were analyzed sequentially [32] by adding sodium sulfite and α-amylase. Analysis of total N was performed using an elemental analyzer (Euro Vector EA3000, EVISA, Milan, Italy) according to the Dumas combustion method [33]. Acid detergent-insoluble crude protein (ADICP) was determined according to Licitra et al. [34]. The anthrone method was used for analysis of water-soluble carbohydrates (WSC) [35]. In vitro dry matter digestibility (IVDMD) was determined according to Goering and Van Soest [36] using ANKOM DaisyII incubator (ANKOM Technologies, Fairport, NY, USA) [37]. Total digestible nutrients were calculated using the equation [88.9 – (0.79 × ADF %)] [38].

Statistical analysis

Data were analyzed by one-way analysis of variance (ANOVA), performed with the general linear model procedure of SPSS Statistics for Windows (version 24.0, IBM, Armonk, NY, USA). The model used for analysis was Yij = μ + Ti + Dj + (T × D)ij + εij, where Yij = observation, µ = mean, Ti = effect of treatments (silage additives), Dj = day of ensiling, (T × D)ij = interaction effect of treatment × day of ensiling, and εij = error term. The experimental unit was the individual ensiling bag. Before analysis, all data were tested for normality and equal variance (t-test); no outliers were identified and the data were normally distributed. If treatment effect was significant at p < 0.05, the mean differences between treatment pairs were determined by Duncan’s multiple range test.


Pre-ensiling characteristics

Heading stage is the optimum stage to forage rye because cude protein (CP) and other nutritive values decline rapidly after this stage [7,39]. Wilting forage rye for 24 h resulted in an increase of DM concentration to 239 g/kg (Table 1). Zhao et al. [9] also noted that the DM concentration was only 165 g/kg when rye was harvested at the heading stage. Therefore, 24-hour wilting could increase DM concentration at a rate of 0.5 percent unit per hour to around 284 g DM/kg. Kim et al. [7] also reported an increase in DM concentration from 178 to 418 g/kg when wilted for 24 h. The wide variations in forage wilting from the other research indicated climate-related factors, namely precipitation, temperature, and wind speed, can significantly influence wilting [40]. Therefore, wilting the rye harvested around this growth stage is advantageous, considering the dry spring weather conditions in Korea.

Table 1. Pre-ensiling characteristics of wilted forage rye after chemical or biological application
Items Treatments1) SEM
Control SDA3 SDA6 LP LB LP + LB
Dry matter (DM, g/kg fresh weight) 239 234 234 247 248 245 1.42
Crude protein (CP, g/kg DM) 198 197 201 198 201 202 1.07
Neutral detergent fiber (NDF, g/kg DM) 525 506 502 531 528 531 2.89
Acid detergent fiber (ADF, g/kg DM) 284 279 280 287 292 294 1.41
Total digestible nutrients (%) 66.4 66.8 66.8 66.2 65.8 65.7 0.11
IVDMD (g/kg DM) 831 851 871 844 833 839 3.26
pH 6.48 6.48 6.21 6.45 6.50 6.45 0.02
Water-soluble carbohydrates (g/kg DM) 143 143 146 150 144 146 0.53
Ammonia-nitrogen (g/kg total N) 3.69 3.64 2.59 3.19 3.23 3.45 0.10
Lactic acid bacteria (Log10 CFU/g fresh weight) 6.20 6.22 6.24 6.89 6.78 6.96 0.08
Total microorganisms (Log10 CFU/g fresh weight) 6.49 6.55 6.49 7.20 7.15 7.42 0.09
Molds (Log10 CFU/g fresh weight) 3.92 3.80 3.48 3.77 3.56 3.56 0.04

Treatments were untreated silage (Control); SDA3, sodium diacetate applied at 3 g/kg fresh weight; SDA6, sodium diacetate applied at 6 g/kg fresh weight; LP, L. plantarum; LB, L. buchneri; LP + LB, L. plantarum + L. buchneri (1:1 ratio). Application rate of inoculants was 1 × 106 CFU/g fresh weight.

ADICP, acid detergent-insoluble crude protein; IVDMD, in vitro dry matter digestibility.

Download Excel Table

Table 1 presents nutrient compositions and microbiological properties of the wilted rye forage treatments before ensiling. The concentrations of NDF and ADF in this experiment demonstrated substantial variation from the other study conducted with forage rye at the heading stage [41], possibly because of the differences in production management, such as seeding rate and harvesting time, as well as climate [42,43].

Consistent with previous reports on early-cut forage rye [5], the wilted forage rye in the present experiment had a substantial WSC concentration (143 g/kg DM). This concentration was greater than the recommended minimum range (60–80 g/kg DM) for lactic acid fermentation [44]. The epiphytic LAB count in the untreated wilted rye before ensiling exceeded the recommended count of 5 Log10 CFU/g to initiate normal silage fermentation [45].

Changes in pH, WSC, and NH3-N during ensiling

The effects of additives and days of ensiling on silage pH, WSC, and NH3-N concentrations of the wilted rye are presented Fig. 2. Fast pH drop occurred at the early stage of ensiling (within five days), and then pH changes flattened throughout the rest of ensiling period (Fig. 2A). The pH of SDA6- and SDA3-treated silages showed steeper drops than LP, LB, and LP + LB. Previous studies on LB inoculation made pH of grass or small-grain silages higher than those of no additives [46], mainly because LB converts lactic acid to other metabolites such as acetic acid, ethanol, and 1, 2-propanediol, which cause the slight rise of the silage pH [47]. Contrarily, we could not confirm this pattern because the pH of the untreated silage was consistently higher than LB-inoculated silages throughout the monitoring period.

Fig. 2. Effects of additives on the dynamics of pH (A), water-soluble carbohydrates (WSC) (B), and ammonia-nitrogen (NH3–N) concentration (C) of wilted rye silage arranged by ensiling days. Application rate of inoculants was 1 × 106 CFU/g wilted weight. Treatments were untreated silage (Control); SDA3, sodium diacetate applied at 3 g/kg wilted weight; SDA6, sodium diacetate applied at 6 g/kg wilted weight; LP, Lactobacillus plantarum; LB, L. buchneri; LP + LB, L. plantarum + L. buchneri(1:1 ratio).
Download Original Figure

The final pH of silage with no additives was 4.10, which is much lower than the value of 5.72 reported by Paradhipta et al. [42] with the untreated forage rye harvested at the dough stage. This emphasizes the importance of harvest maturity for silage fermentation of forage rye. Changes in DM and WSC in winter forages of different maturity can influence microbial growth and silage fermentation [25].

The decline of WSC concentration occurred in all silage treatments, which was rapid during the first 5 days of ensiling. This pattern matched to the pH declines (Fig. 2B). Silages inoculated with LP, LB or LP + LB exhibited a faster rate of WSC decline during the first 5 days of fermentation than other treatments. Approximately 80% of the WSC in the wilted rye was utilized within 5 days of ensiling in LB, LP, and LP + LB treatments, which was significantly greater than other treatments. After 45 days, the residual WSC concentration was the greatest in the untreated and SDA6-treated silages. This suggests greater WSC utilization by the microbial inoculants. Decline of WSC during ensiling is usually due to conversion of fermentable sugars into organic acids, predominantly lactic acid by lactic fermenting bacteria [6]. Alternatively, WSC could also feed undesirable enterobacteria, clostridia, acetobacter bacteria, mold, and yeast, which results in a low lactic acid concentration silage. The insufficient acidify could not prevent the undesirable bacteria growth [6]. In this experiment, the faster decline of WSC in lactic bacteria inoculated treatments indicated higher lactic acid concentration and lower silage pH.

In all silages, the NH3-N concentration increased gradually as fermentation proceeded, and stabilized to the final phases of ensiling (Fig. 2C). The degree of NH3-N increase was generally greater in the untreated silage than those of treatments. At day 45 of ensiling, the NH3–N concentration was lowest in SDA6-treated and LP-inoculated silages and highest in the untreated silage. The protein degradation into various nitrogenous compounds during ensiling is inevitable. Especially, NH3-N is low in the nutritional value [48]. The formation of NH3-N is accelerated by enhanced activity of microorganisms that degrade true protein fractions into ammonia [6]. The NH3-N concentration should be below 100 g/kg total N in silages to be considered as desirable fermentation [6]. The NH3-N values in this study indicated that severe protein degradations did not occur during ensiling.

Within the early- days of ensiling, proteolysis occurred actively. The increase of NH3-N formation was slowest when treated with SDA6 and LP. At the early of the ensiling, a sharp decline of silage pH reduced enzyme activity and microbial proteolysis [4951]. Protease begins to decrease its activity as the pH declines, with approximately 67% of the enzyme activity being lost within 24 h at the pH ranging from 4 to 5 [52]. Despite the lower silage pH of the LP + LB-treated silage than SDA6-treated silage, the NH3–-N concentration was relatively higher during the ensiling process of LP + LB-treated silage, possibly because of the antimicrobial properties at 6g SDA per kg wilted forage rye, which may be sufficient to suppress the aerobic microorganisms activities and enzymes involved proteolysis [48]. Zhao et al. [9] reported a substantially higher NH3–N concentration and higher pH of wilted rye than our study. The initial WSC concentration has been identified as a prerequisite for accelerating the pH decline, which is also related to lower NH3–N concentrations [6,53]. In this study, lactic acid concentration was higher and silage pH was lower than Zhao et al. [9], indicating the higher WSC boosted lactic acid fermentation and lead to a lower silage pH, decreasing protein degradation.

Organic acid changes during ensiling

Lactic acid formation increased gradually with ensiling, with the highest increase rates within the first 5 days of fermentation (Fig. 3A). From day 0 to 3 of ensiling, lactic acid production was the highest in the LP treated silage, followed by LP + LB or LB, and the lowest amount with the SDA-treated silages. Lactic acid concentration in the SDA-treated silages increased slower than the inoculant treatments. Lactic acid accumulation reached its plateau between 10 and 20 ensiling days, and the concentrations in LP and LP + LB were the highest among the treatments at day 45 of ensiling. Lactic acid concentrations in the experimental silages exceeded the typical ranges (20–40 g/kg DM), because of the high moisture content of the wilted rye at ensiling (650–750 g/kg) promoting lactic acid formation during ensiling [54]. Previous research identified LP as a LAB strain whose main end-product is lactate, which is mainly involved in the rapid acidifications of silage after ensiling, resulting in early suppression of unwanted microorganisms and some fermentation end-products [23,55].

Fig. 3. Effects of additives on organic acid content of wilted rye silage arranged by ensiling days; lactic acid (A), acetic acid (B), and lactic acid: acetic acid (C). Application rate of inoculants was 1 × 106 cfu/g wilted weight. Treatments were untreated silage (Control); SDA3, sodium diacetate applied at 3 g/kg wilted weight; SDA6, sodium diacetate applied at 6 g/kg wilted weight; LP, Lactobacillus plantarum; LB, L. buchneri; LP + LB, L. plantarum + L. buchneri (1:1 ratio).
Download Original Figure

Acetic acid presented in the highest concentration when treated with SDA at the first day of ensiling (Fig. 3B). From day 2 to 5 of ensiling, acetic acid concentration increased gradually followed by a slight reduction, until no significant changes to the end of ensiling period. Acetic acid concentration in the LB-treated silages maintained at the typical range of 3%–4% as the previous report [54]. The substantially high acetic acid formation in LB-treated silage could be explained with the promotion of heterofermentative metabolism in this treatment [20,24,56]. Acetic acid concentration in this study was lowest in the LP-treated silage, which is consistent with the findings of Auerbach and Theobald [5], who reported higher lactic acid but lower acetic acid concentrations by LP inoculation.

All silage treatments showed an increase in the lactic acid to acetic acid ratio during the early phase of ensiling, which then decreased slightly until stabilized (Fig. 3C). This ratio was generally higher for the LP- and LP + LB-treated silages, reaching its maximum value after 5 days of ensiling. The untreated and LB-treated silages reached their peak ratio after 10 days of ensiling, while the SDA-treated silages reached their peak ratio after 20 days of fermentation. At day 45 of ensiling, the highest ratio (5.76) was seen for the LP-treated silages, while lower ratios were observed in SDA6- and LB-treated silages. A lactic acid to acetic acid ratio higher than 3 indicates dominant homolactic fermentation [49], and all the treatments except for SDA6 and LB demonstrated the ratio higher than 3.0.

Microbial population changes during ensiling

In all silages, LAB multiplied rapidly and generally became dominant within the first 5 days of ensiling, especially in the inoculated silages, indicating that LAB was dominant in the fermentation microbial community (Fig. 4A). Generally, LAB counts reached their maximum on days 5–10 of ensiling, followed by a gradual decrease. For example, LAB counts in the untreated and SDA3-treated silages reached their maximum after 10 days of ensiling. At day 45 of ensiling, LAB counts were highest in the LB-treated silages and lowest in the untreated and SDA3-treated silages. The total microorganism population followed the same pattern as the LAB count throughout the fermentation process (Fig. 4B). Mold count declined with progression of ensiling, reaching an undetectable level in the additive-treated silages after 20 days of ensiling (Fig. 4C). Mold became undetectable after 45 days of ensiling in the untreated silage. However, molds reached to undetectable count level faster (day 10) in the silages treated with LP, LP + LB, or SDA6. In these silages, pH reduction to below 4 occurred within 5–10 days of ensiling (Fig. 2A). At low silage pH (~4), a greater proportion of acetate molecule exists in an undissociated form, which can easily penetrate the cell membrane of yeasts and molds and release hydrogen ions into the cytosol. This increases the ATP expenditures of the organism needed to maintain homeostasis, thus disrupting cellular metabolism [17,57].

Fig. 4. Effects of additives on the dynamics of microbial population in wilted rye silage arranged by ensiling days; lactic acid bacteria (A), total microorganisms (B), and mold (C). Application rate of inoculants was 1 × 106 CFU/g wilted weight. Treatments were untreated silage (Control); SDA3, sodium diacetate applied at 3 g/kg wilted weight; SDA6, sodium diacetate applied at 6 g/kg wilted weight; LP, Lactobacillus plantarum; LB, L. buchneri; LP + LB, L. plantarum + L. buchneri (1:1 ratio).
Download Original Figure

Yuan et al. [14] reported that SDA applied at 7 g/kg fresh weight had an inhibitory effect on both undesirable microorganisms and LAB development. This study also confirmed lower lactic acid production and lower count of LAB than other treatments when SDA was applied at 6 g/kg wilted rye. Wen et al. [16] also observed an initial delay in lactic acid formation compared to control silage when alfalfa (Medicago sativa L.) forage was treated with SDA (7 g/kg fresh weight), which was ascribed to the suppressive effect of SDA on LAB viability. The progressive decline of LAB with longer ensiling durations is likely linked to the excessively low silage pH and exhaustion of WSC, both of which suppress LAB growth [6,58]. Low silage pH suppresses the dominance of LAB, when the pH reaches 4.0 [50,59]. These could well explain the decline of LAB population in the later stage of ensiling in this experiment.

Nutrient value changes during ensiling

There was a gradual decline in DM concentration over the course of the ensiling process (Fig. 5A), likely because of the loss of organic matter to water and carbon dioxide under anaerobic conditions [6,58].

Fig. 5. Effects of additives on nutritive characteristics of wilted rye silage arranged by ensiling days; dry matter (A), crude protein (B), and ADICP (C). Application rate of inoculants was 1 × 106 CFU/g wilted weight. Treatments were untreated silage (Control); SDA3 = sodium diacetate applied at 3 g/kg wilted weight; SDA6, sodium diacetate applied at 6 g/kg wilted weight; LP, Lactobacillus plantarum; LB, L. buchneri; LP + LB, L. plantarum + L. buchneri (1:1 ratio); ADICP, acid detergent-insoluble crude protein.
Download Original Figure

The decrease in CP concentration during the first 3 days of ensiling in this study (Fig. 5B) could be ascribed to the increased proteolysis caused by the activity of plant enzymes and existing microorganisms in the initial phases of ensiling [6,54,60]. Ammonia is a volatile compound and degradation of protein fractions into NH3–N, which particularly increased in the first 3 days of ensiling, may possibly explain the decrease of CP concentration [6]. After day 45 of ensiling, SDA6-treated silages had the greatest CP concentration while the lowest NH3–N concentration, implying lower loss of the protein fractions as ammonia by the microbial proteolysis. The lower NH3–N concentration in SDA6-treated silage indicated that SDA had significant effect in preserving forage proteins throughout ensiling. After 45 days, the average ADICP concentration in all silages was 76.5 g/kg CP (Fig. 5C), which is near the threshold of 75 g/kg CP reflecting normal silage fermentation [61]. The ADICP demonstrated significant variations among the treatments around day 20 and the variation in ADICP became narrow to a 3% range in the later phase of ensiling. A high concentration of ADICP is indicative of heat-damaged protein, which has low nutritional value for animals [62].

The effects of the additives and days of ensiling on TDN and IVDMD of the silages are presented in Table 2. The silages demonstrated inconsistency in TDN and IVDMD among the treatment across the progress of ensiling period.

Table 2. Effect of different additives on the dynamic change of TDN and IVDMD of wilted forage rye sampled at days of ensiling
Items Additives1) Days of ensiling SEM
1 2 3 5 10 20 30 45
TDN (%) Control 66.5gB 67.3efBC 67.3efBC 67.6cdeC 68.1aC 67.6bcdD 67.7bcBC 67.1fC 0.10
SDA3 67.2eA 67.9cdA 67.7dAB 68.5abA 68.7aB 68.8aA 68.2bcA 67.9cdAB 0.11
SDA6 67.4dA 67.8cA 68.0bcA 68.3bAB 69.5aA 67.8cCD 68.2bA 68.2bA 0.12
LP 66.5dB 67.0cC 67.0cCD 68.2aB 68.0aC 67.7bD 66.8cdD 66.7cdD 0.13
LB 67.4bcA 67.1cdC 66.9dD 67.5bC 68.1aC 68.0aBC 67.9aAB 67.9aAB 0.09
LP + LB 67.2dA 67.5cB 67.6cAB 67.6cC 68.6aB 68.2bB 67.4cdC 67.7cB 0.09
IVDMD (g/kg DM) Control 836bB 829cdC 860aA 827dCD 814eC 797fgD 835bA 795gD 4.20
SDA3 839bcB 843bB 847bB 843bAB 825dB 859aA 828dAB 823dB 2.54
SDA6 864aA 855bA 870aA 851bA 847bcA 849bB 836dA 840cdA 2.33
LP 824bcC 837aB 831abC 841aAB 790dE 823bcC 819cBC 817cC 3.16
LB 823abC 824abCD 826aC 824aD 817bBC 805cD 816bC 816bC 1.47
LP + LB 823bC 819bD 834aC 835aBC 798cD 826abC 819bBC 817bC 2.41

Treatments were untreated silage (Control); SDA3, sodium diacetate applied at 3 g/kg fresh weight; SDA6, sodium diacetate applied at 6 g/kg fresh weight; LP, L. plantarum; LB, L. buchneri; LP + LB, L. plantarum + L. buchneri (1:1 ratio). Application rate of inoculants was 1 × 106 cfu/g fresh weight.

Within rows, means with dissimilar superscripts differ (p < 0.05).

Within each column, means with dissimilar superscripts differ (p < 0.05).

TDN, total digestible nutrients; IVDMD, in vitro dry matter digestibility; DM, dry matter.

Download Excel Table

At the end of ensiling, the average IVDMD was highest in the silage treated with SDA6 (840 g/kg DM), followed by SDA3, and lactic bacteria inoculation treatments. The untreated silage was lowest. The difference in IVDMD across the silages treated with microbial inoculants was not significant (mean of 817 g/kg DM). There must be decrease of non-structural carbohydrates and this reduction resulted in the relative increase of structural carbohydrate proportions, thereby lowering TDN and IVDMD as ensiling progresses [63]. Some reports proposed possible hydrolysis of fibrous carbohydrates due to the impact of accumulated acidifying agents during ensiling fermentation [6,64], however, the inconsistency of the data do not support the theory.


Although existing conditions of the wilted rye may be sufficient to achieve lactic acid fermentation, the application of fermentation aids will carry specific benefits when the cool-season annual forage crop produces a large amount of wet biomass. As confirmed in this study, 24-hour wilting is marginally enough for moisture control. Also, the chances of unfavorable weather conditions warrant the application of silage additives to achieve enhanced forage preservation. For example, the application of SDA demonstrated its effectiveness in suppressing overall microbial activities while homolactic bacteria inoculation may help to boost lactic fermentation of the forage. Silage additives must be applied considering variable harvest environment and storage conditions.

Competing interests

No potential conflict of interest relevant to this article was reported.

Funding sources

This research was supported by Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ01621302).


Not applicable.

Availability of data and material

Upon reasonable request, the datasets of this study can be available from the corresponding author.

Authors’ contributions

Conceptualization: Li YF, Kim JG.

Data curation: Li YF.

Formal analysis: Wang LL, Kim HJ.

Methodology: Li YF, Kim JG.

Software: Jeong EC.

Validation: Ahmadi F.

Investigation: Li YF.

Writing - original draft: Li YF.

Writing - review & editing: Li YF, Jeong EC, Wang LL, Kim HJ, Ahmadi F, Kim JG.

Ethics approval and consent to participate

This article does not require IRB/IACUC approval because there are no human and animal participants.



Kaspar TC, Radke JK, Laflen JM. Small grain cover crops and wheel traffic effects on infiltration, runoff, and erosion. J Soil Water Conserv. 2001; 56:160-4


Kennelly JJ, Weinberg ZG. Small grain silage.In In: Buxton DR, Muck RE, Harrison JH, editors.editors Silage science and technology. Madison, WI: American Society of Agronomy, Crop Science Society of America, Soil Science Society of America. 2003; p p. 749-79


Moore EB, Wiedenhoeft MH, Kaspar TC, Cambardella CA. Rye cover crop effects on soil quality in no-till corn silage–soybean cropping systems. Soil Sci Soc Am J. 2014; 78:968-76


Kumssa TT, Anderson JD, Butler TJ, Ma XF. Small grains as winter pasture in the Southern Great Plains of the United States.In In: Kindomihou VM, editor.editor Grasses and grassland aspects. London: IntechOpen. 2019


Auerbach H, Theobald P. Additive type affects fermentation, aerobic stability and mycotoxin formation during air exposure of early-cut rye (Secale cereale L.) silage. Agronomy. 2020; 10:1432


McDonald P, Henderson N, Heron S. The biochemistry of silage. Marlow: Chalcombe Publications. 1991


Kim JG, Chung ES, Seo S, Ham JS, Kang WS, Kim DA. Effects of maturity at harvest and wilting days on quality of round baled rye silage. Asian-Australas J Anim Sci. 2001; 14:1233-7


Borreani G, Tabacco E, Schmidt RJ, Holmes BJ, Muck RE. Silage review: factors affecting dry matter and quality losses in silages. J Dairy Sci. 2018; 101:3952-79


Zhao GQ, Wei SN, Liu C, Kim HJ, Kim JG. Effect of harvest dates on β-carotene content and forage quality of rye (Secale cereale L.) silage and hay. J Anim Sci Technol. 2021; 63:354-66


Kalúzová M, Juráček M, Bíro D, Gálik B, Šimko M, Rolinec M, et al. The effect of biological additive on the fermentation quality of whole-crop rye silage. Acta Fytotech Zootech. 2021; :24


Arriola KG, Vyas D, Kim D, Agarussi MCN, Silva VP, Flores M, et al. Effect of Lactobacillus hilgardii, Lactobacillus buchneri, or their combination on the fermentation and nutritive value of sorghum silage and corn silage. J Dairy Sci. 2021; 104:9664-75


Bruckner PL, Hanna WW. In vitro digestibility of fresh leaves and stems of small-grain species and genotypes. Crop Sci. 1990; 30:196-202


Yitbarek MB, Tamir B. Silage additives: review. Open J Appl Sci. 2014; 4:258-74


Yuan XJ, Wen AY, Desta ST, Wang J, Shao T. Effects of sodium diacetate on the fermentation profile, chemical composition and aerobic stability of alfalfa silage. Asian-Australas J Anim Sci. 2017; 30:804-10


Muck RE. Silage microbiology and its control through additives. Rev Bras Zootec. 2010; 39:183-91


Wen AY, Yuan X, Wang J, Desta ST, Shao T. Effects of four short-chain fatty acids or salts on dynamics of fermentation and microbial characteristics of alfalfa silage. Anim Feed Sci Technol. 2017; 223:141-8


Danner H, Holzer M, Mayrhuber E, Braun R. Acetic acid increases stability of silage under aerobic conditions. Appl Environ Microbiol. 2003; 69:562-7


Kleinschmit DH, Schmidt RJ, Kung L. The effects of various antifungal additives on the fermentation and aerobic stability of corn silage. J Dairy Sci. 2005; 88:2130-9


Muck RE, Nadeau EMG, McAllister TA, Contreras-Govea FE, Santos MC, Kung L. Silage review: recent advances and future uses of silage additives. J Dairy Sci. 2018; 101:3980-4000


Ranjit NK, Kung L. The effect of Lactobacillus buchneri, Lactobacillus plantarum, or a chemical preservative on the fermentation and aerobic stability of corn silage. J Dairy Sci. 2000; 83:526-35


Ávila CLS, Carvalho BF, Pinto JC, Duarte WF, Schwan RF. The use of Lactobacillus species as starter cultures for enhancing the quality of sugar cane silage. J Dairy Sci. 2014; 97:940-51


Filya I. The effect of Lactobacillus buchneri, with or without homofermentative lactic acid bacteria, on the fermentation, aerobic stability and ruminal degradability of wheat, sorghum and maize silages. J Appl Microbiol. 2003; 95:1080-6


Adesogan AT, Krueger N, Salawu MB, Dean DB, Staples CR. The influence of treatment with dual purpose bacterial inoculants or soluble carbohydrates on the fermentation and aerobic stability of bermudagrass. J Dairy Sci. 2004; 87:3407-16


Auerbach H, Theobald P, Kroschewski B, Weiss K. Effects of various additives on fermentation, aerobic stability and volatile organic compounds in whole-crop rye silage. Agronomy. 2020; 10:1873


Hu W, Schmidt RJ, McDonell EE, Klingerman CM, Kung L. The effect of Lactobacillus buchneri 40788 or Lactobacillus plantarum MTD-1 on the fermentation and aerobic stability of corn silages ensiled at two dry matter contents. J Dairy Sci. 2009; 92:3907-14


Oliveira AS, Weinberg ZG, Ogunade IM, Cervantes AA, Arriola KG, Jiang Y, et al. Meta-analysis of effects of inoculation with homofermentative and facultative heterofermentative lactic acid bacteria on silage fermentation, aerobic stability, and the performance of dairy cows. J Dairy Sci. 2017; 100:4587-603


Santos EM, Pereira OG, Garcia R, Ferreira CLLF, Oliveira JS, Silva TC. Effect of regrowth interval and a microbial inoculant on the fermentation profile and dry matter recovery of guinea grass silages. J Dairy Sci. 2014; 97:4423-32


Wei SN, Li YF, Jeong EC, Kim HJ, Kim JG. Effects of formic acid and lactic acid bacteria inoculant on main summer crop silages in Korea. J Anim Sci Technol. 2021; 63:91-103


Broderick GA, Kang JH. Automated simultaneous determination of ammonia and total amino acids in ruminal fluid and in vitro media. J Dairy Sci. 1980; 63:64-75


Zhao GQ, Wei SN, Li YF, Jeong EC, Kim HJ, Kim JG. Comparison of forage quality, productivity and β-carotene content according to maturity of forage rye (Secale cereale L.). J Korean Soc Grassl Forage Sci. 2020; 40:123-30


Madigan MT, Martinko JM, Brock TD. Brock biology of microorganisms. Upper Saddle River, NJ: Pearson Prentice Hall. 2006


Van Soest PJ, Robertson JB, Lewis BA. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J Dairy Sci. 1991; 74:3583-97


Jean-Baptiste-André Dumas. Science. 1884; 3:750-2


Licitra G, Hernandez TM, Van Soest PJ. Standardization of procedures for nitrogen fractionation of ruminant feeds. Anim Feed Sci Technol. 1996; 57:347-58


Yemm EW, Willis AJ. The estimation of carbohydrates in plant extracts by anthrone. Biochem J. 1954; 57:508-14


Goering HK, Van Soest PJ. Forage fiber analyses: apparatus, reagents, procedures, and some applications. Agricultural Handbook No. 379 Washington, DC: US Department of Agriculture-Agricultural Research Service (USDA-ARS). 1970


Wei SN, Jeong EC, Li YF, Kim HJ, Ahmadi F, Kim JG. Evaluation of forage production, feed value, and ensilability of proso millet (Panicum miliaceum L.). J Anim Sci Technol. 2022; 64:38-51


Schroeder JW. Forage nutrition for ruminants. FargoNorth Dakota State University 2004


Geren H. Dry matter yield and silage quality of some winter cereals harvested at different stages under Mediterranean climate conditions. Turk J Field Crops. 2014; 19:197-202


Marsh R. The effects of wilting on fermentation in the silo and on the nutritive value of silage. Grass Forage Sci. 1979; 34:1-10


Srigopalram S, Ilavenil S, Kuppusamy P, Yoon YH, Kim WH, Choi KC. Silages of rye harvested at different stages: a study on microbial inoculants responses in improving rye silage fermentation quality. J Korean Grassl Forage Sci. 2017; 37:189-94


Paradhipta DHV, Joo YH, Lee HJ, Lee SS, Kwak YS, Han OK, et al. Effects of wild or mutated inoculants on rye silage and its rumen fermentation indices. Asian-Australas J Anim Sci. 2020; 33:949-56


Lee SS, Choi JS, Paradhipta DHV, Joo YH, Lee HJ, Noh HT, et al. Application of selected inoculant producing antifungal and fibrinolytic substances on rye silage with different wilting time. Processes. 2021; 9:879


Woolford MK. The silage fermentation. New York: Marcel Dekker. 1984; p p. 350


Kung L, Muck R. Effects of silage additives on ensiling. In: Proceedings from the Silage: Field to Feedbunk, North American Conference. 1997; Hershey, PA. p p. 187-99


Kleinschmit DH, Kung L. A meta-analysis of the effects of Lactobacillus buchneri on the fermentation and aerobic stability of corn and grass and small-grain silages. J Dairy Sci. 2006; 89:4005-13


Oude Elferink SJWH, Krooneman J, Gottschal JC, Spoelstra SF, Faber F, Driehuis F. Anaerobic conversion of lactic acid to acetic acid and 1,2-propanediol by Lactobacillus buchneri. Appl Environ Microbiol. 2001; 67:125-32


Yuan X, Wen A, Desta ST, Dong ZH, Shao T. Effects of four short-chain fatty acids or salts on the dynamics of nitrogen transformations and intrinsic protease activity of alfalfa silage. J Sci Food Agric. 2016; 97:2759-66


Kung L, Stokes MR, Lin CJ. Silage additives.In In: Buxton DR, Muck RE, Harrison JH, editors.editors Silage science and technology. Madison, WI: American Society of Agronomy, Crop Science Society of America, Soil Science Society of America. 2003; p p. 305-60


Seglar B. Fermentation analysis and silage quality testing.In Minnesota Dairy Health Conference. 2003; St. Paul. MN: p p. 119-35


Wilkinson JM, Davies DR. The aerobic stability of silage: key findings and recent developments. Grass Forage Sci. 2013; 68:1-19


Jones BA, Hatfield RD, Muck RE. Characterization of proteolysis in alfalfa and red clover. Crop Sci. 1995; 35:537-41


Heron SJE, Edwards RA, Phillips P. Effect of pH on the activity of ryegrass Lolium multiflorum proteases. J Sci Food Agric. 1989; 46:267-77


Kung L, Shaver RD, Grant RJ, Schmidt RJ. Silage review: interpretation of chemical, microbial, and organoleptic components of silages. J Dairy Sci. 2018; 101:4020-33


Kung L, Shaver R. Interpretation and use of silage fermentation analysis reports. Focus Forage. 2001; 3:1-5


Driehuis F, Oude Elferink SJWH, Spoelstra SF. Anaerobic lactic acid degradation during ensilage of whole crop maize inoculated with Lactobacillus buchneri inhibits yeast growth and improves aerobic stability. J Appl Microbiol. 1999; 87:583-94


Drouin P, Tremblay J, Chaucheyras-Durand F. Dynamic succession of microbiota during ensiling of whole plant corn following inoculation with Lactobacillus buchneri and Lactobacillus hilgardii alone or in combination. Microorganisms. 2019; 7:595


Kim JS, Lee YH, Kim YI, Ahmadi F, Oh YK, Park JM, et al. Effect of microbial inoculant or molasses on fermentative quality and aerobic stability of sawdust-based spent mushroom substrate. Bioresour Technol. 2016; 216:188-95


Woolford MK. Microbiological screening of the straight chain fatty acids (C1-C12) as potential silage additives. J Sci Food Agric. 1975; 26:219-28


Henderson N. Silage additives. Anim Feed Sci Technol. 1993; 45:35-56


Chamberlain AT, Wilkinson JM. Feeding the dairy cow. 2nd ed Lincoln: Chalcombe Publications. 2000


Kaiser AG, Piltz JW, Burns HM, Griffiths NW. Top fodder. In: Successful silage. 2nd ed Orange, NSW: Dairy Australia and New South Wales Department of Primary Industries. 2004


Gomes ALM, Jacovaci FA, Bolson DC, Nussio LG, Jobim CC, Daniel JLP. Effects of light wilting and heterolactic inoculant on the formation of volatile organic compounds, fermentative losses and aerobic stability of oat silage. Anim Feed Sci Technol. 2019; 247:194-8


Desta ST, Yuan XJ, Li J, Shao T. Ensiling characteristics, structural and nonstructural carbohydrate composition and enzymatic digestibility of Napier grass ensiled with additives. Bioresour Technol. 2016; 221:447-54


Korean Meteorological Administration. Climate satistics analysis [Internet]. 2022 [Cited 2022 Nov 17].