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ISSN : 2287-5824(Print)
ISSN : 2287-5832(Online)
Journal of The Korean Society of Grassland and Forage Science Vol.44 No.4 pp.226-239
DOI : https://doi.org/10.5333/KGFS.2024.44.4.226

Fermentative Characteristics in Italian Ryegrass Silage by Multiple Probiotic Inoculants Based on Lactiplantibacillus plantarum

Young Sang Yu1, Yan Fen Li1, Xaysana Panyavong1, Li Zhunang Wu1, Jeong Ung Hwang1, Li Li Wang2, Jong Geun Kim1,2*
1Graduate School of International Agricultural Technology, SNU, Pyeongchang 25354, Korea
2Research Institute Eco-Friendly Livestock Science, GreenBio Science and Technology, SNU, Pyeongchang 25354, Korea
* Corresponding author: Jong Geun Kim, Graduate School of International Agricultural Technology and GBST, Seoul National University, Pyeongchang 25354, Korea, Tel: +82-33-339-5728, E-mail: forage@snu.ac.kr
September 19, 2024 October 21, 2024 October 25, 2024

Abstract


Silage inoculants, which include beneficial microorganisms like lactic acid bacteria (LAB), play a vital role in modern silage production by enhancing fermentation quality. This study evaluated the effectiveness of various commercial inoculants on the fermentation dynamics of Italian ryegrass silage over 45 days. The treatments included a control group and five inoculant formulations: T1 (Lactiplantibacillus plantarum), T2 (Lactiplantibacillus plantarum and Pediococcus pentosaceus), T3 (Lactiplantibacillus plantarum and Pediococcus pentosaceus and Lactiplantibacillus buchneri), T4 (Lactiplantibacillus plantarum and Lactiplantibacillus acidophilus and Lactiplantibacillus bulgaricus), and T5 (Lactiplantibacillus plantarum and Pediococcus pentosaceus and Enterococcus faecium). After 45 days, all treatment groups exhibited significantly higher crude protein (CP) content compared to the control group (80.64 g/kg dry matter (DM), p<0.05). Treatments T2 and T5, which incorporated combinations of Lactiplantibacillus plantarum, Pediococcus pentosaceus and Enterococcus faecium, showed higher CP contents at 105.53 and 107.05 g/kg DM, respectively. The inoculated silages also demonstrated a rapid pH reduction within the early days, with Lactiplantibacillus plantarum in T1 reducing the pH to 4.0 within four days. Additionally, inoculated treatments had significantly higher lactic acid levels than the control (67.96 g/kg DM, p<0.05), and T3 (Lactiplantibacillus buchneri) produced higher acetic acid levels (16.07 g/kg DM, p<0.05) than other inoculants. The control group also had a notably higher ammonia nitrogen content. In conclusion, while single-strain inoculants like Lactiplantibacillus plantarum are effective for rapid acidification, the use of combined bacterial strains can further enhance silage quality by improving lactic acid fermentation and nutrient preservation, particularly in treatments like Lactiplantibacillus plantarum and Pediococcus pentosaceus and Lactiplantibacillus buchneri and Enterococcus faecium.


Fermentation , Inoculant , Lactic acid bacteria , Quality , Silage , Italian ryegrass

초록

    Ⅰ. INTRODUCTION

    Silage production is a critical aspect of modern livestock farming, providing a stable and nutritious feed source throughout the year. Ensiling, the process of fermenting forage under anaerobic conditions, is enhanced by the use of silage inoculants. These microbial additives, primarily composed of lactic acid bacteria (LAB), are strategically applied to accelerate the fermentation process and improve the overall quality of the silage (Bai et al., 2021). The key function of these inoculants is to drive the conversion of water-soluble carbohydrates into lactic acid, a process that not only lowers the pH but also inhibits the growth of undesirable microorganisms, thereby preserving the forage (Kung, 2018).

    While homolactic fermentation, driven by LAB, is well-known for its efficiency in reducing pH and minimizing dry matter losses (Li et al., 2022), recent advancements in microbial technology have introduced a variety of inoculant formulations (Lobo et al., 2019). These formulations often combine multiple bacterial strains to target specific aspects of the fermentation process, aiming to optimize both the speed and quality of fermentation. However, despite the availability of numerous commercial products, there is a need for comprehensive studies that evaluate their comparative efficacy under consistent conditions (Gonda et al., 2023).

    This study seeks to address this gap by comparing the effectiveness of various silage inoculants from different manufacturers, focusing on their impact on the fermentation process and quality of Italian ryegrass silage over a 45-day ensiling period. By examining the fermentation dynamics and resulting nutritional profiles, this research aims to provide insights that can guide the selection of inoculants best suited for enhancing silage production.

    Ⅱ. MATERIALS AND METHODS

    1. Experimental design

    This experiment was conducted at the experimental field of Pyeongchang Campus, Seoul National University, Gangwon-do, Republic of Korea (Latitude: 37° 32' 46.10" N, Longitude: 128° 26' 17.90" E). The Italian ryegrass (IRG) was seeded in the “Kowinearly” variety, developed by the National Institute of Animal Science (Choi et al., 2011) on October 6, 2022, and harvested from the heading stage on May 18, 2023. After harvest with manual mixing, the IRG was chopped into 2-3 cm by a cutter (SC-7000, Agricultural Machinery, Inc., Daegu, South Korea), and then treated with silage inoculant. The composition of the treatment product is as follows: (control): 100 mL distilled water /50 kg of fresh matter, (T1): Lactiplantibacillus (Lactobacillus) plantarum at the rate of 3×105 cfu/50 kg, (T2): Lactiplantibacillus plantarum and Pediococcus pentosaceus at the rate of 2×104 cfu/50 kg, (T3): Lactiplantibacillus plantarum and Pediococcus pentosaceus and Lactiplantibacillus buchneri at the rate of 2.5×105 cfu/50 kg, (T4): Lactiplantibacillus plantarum and Lactiplantibacillus acidophilus and Lactiplantibacillus bulgaricus at the rate of 4.25×103 cfu/50 kg, (T5): Lactiplantibacillus plantarum and Pediococcus pentosaceus and Enterococcus faecium at the rate of 5×105 cfu/50 kg. All additives were produced by different companies. Approximately 300g treated material was packed into plastic vacuum bags, and immediately sealed vacuum packer (GOV-4040, Gaonpack Co., Gyeonggi-do, Korea). Treatments were replicated 3 times. The silages opened after 1, 2, 3, 4, 10, 20, 30 and 45 days.

    2. Analysis of fermentative characteristics

    To measure acidity, 10 g of fresh silage from each treatment were placed in conical flasks with 90 mL of distilled water, sealed, shaken for an h, and stored in a refrigerator at 4℃ for 24 h. The mixture was then filtered, and the pH was measured using a pH meter (AB 150, Fisher Scientific International, Inc., Pittsburgh, US). Silage extracts were stored at -20℃ for further analysis of volatile fatty acids, lactic acid, and ammonia nitrogen. For organic acid analysis, thawed extracts were centrifuged, filtered, and analyzed using HPLC equipped with a refractive index detector. The column employed was the Agilent Hi-Plex H (7.7 x 300 mm, 8 μm, part number PL1170-6830); Mobile phase: 0.005 M H2SO4; Flow rate: 0.7 mL/min). The column temperature was maintained at 60℃, and the pressure was set at 4.6 MPa (46 bar, 670 psi). Detection of the relative volatility (RV) occurred at 55℃.

    Ammonia nitrogen concentration was determined using a modified phenol-hypochlorite procedure as outlined by Broderick and Kang (1980). The thawed extract was mixed thoroughly under natural environmental conditions. Subsequently, 12 mL of the extract were transferred into a 15 mL centrifuge tube using a pipette and then centrifuged at 3000 rpm, maintaining a temperature of 4℃ for 15 min. A 0.02 mL aliquot of the supernatant was injected into a 25 mL test tube, followed by the sequential addition of 1 mL of phenol reagent and 1 mL of alkali-hypochlorite reagent. The test tube was immediately covered and shaken vigorously to ensure thorough mixing of the reagents and supernatant. The test tube was then placed in a 37℃ water bath for 15 min. to allow for color reaction. Following that, 8 mL of distilled water were added, and the mixture and combination was vortexed thoroughly to ensure proper mixing. The spectrophotometric analysis was carried out at 630 nm wavelength using a Libra S70 Double Beam Spectrophotometer (Biochrom, Korea). Calibration at 630 nm was performed using a blank, and the ammonia nitrogen concentration of both standard and sample extractions was measured.

    3. Analysis of nutritional values

    Fresh Italian ryegrass samples taken before ensiling and silage samples obtained upon opening were sub-sampled for the analysis of dry matter (DM), crude protein (CP), acid detergent fiber (ADF), neutral detergent fiber (NDF), water-soluble carbohydrates (WSC), and in vitro dry matter digestibility (IVDMD). All chemical analyses were conducted and reported on a dry matter basis. Upon opening the polyethylene bags containing silage, the samples were meticulously mixed, and approximately 300 g of each sample was extracted.

    The dry matter analysis proceeded as follows: The samples were first placed in paper bags, weighed, and then subjected to air-forced drying in an oven at 65℃ for 72 h. After the drying process, all samples were ground to pass through a 1 mm screen using the "Wiley Mill 4 1/2 Horse Power Unit Motor" instrument (Thomas Scientific, Inc., New Jersey, USA). Following grinding, the ground samples were transferred into plastic bottles and stored in a dried storage room at 4℃ until further analysis.

    The crude protein (CP) analysis was conducted using the Dumas method, as originally described by Dumas (1831). Before sampling, the contents of the plastic bottle were meticulously mixed to minimize errors. Dried sample powder ranging from 9 mg to 11 mg was accurately weighed and enclosed in tin foil, where it was compressed into small squares for precise protein content analysis. The CP analysis was performed using the "Automatic Vector Analyzer Euro Vector EA3000" instrument (EVISA Co., Ltd, Milan, Italy), adhering to established protocols and methodologies.

    For neutral detergent fiber (NDF) and acid detergent fiber (ADF) analysis, 0.5 ~ 0.6 g of dried sample powder was precisely weighed and enclosed in Nylon filter bags (50 mm × 55 mm, ANKOM F57, ANKOM Tech., Fairport, NY). In the NDF procedure, 20 g of sodium sulfate and 12 mL of alpha-amylase were added. However, these additions were not necessary for ADF analysis. The NDF and ADF analyses were conducted following the methods outlined by Van Soest et al. (1991) utilizing the ANKOM A2000 Automated Fiber Analyzer (Ankom Technologies, Inc., Fairport, NY, USA).

    The determination of water-soluble carbohydrates (WSC) in oven-dried materials was conducted using a modified Anthrone-Sulfuric acid colorimetry method as described by Yemm and Willis (1954). Ground samples weighing 0.2 g from each treatment were taken in triplicate and placed in labeled 250 mL conical flasks with 200 mL distilled water, securely sealed, and shaken using a shaker (Green Sseriker, Vision Scientific, Korea) for one hour. The mixture was subsequently filtered through (Whatman No. 1, AVANTEC).

    Next, 2 mL of the filtrate was pipetted into labeled test tubes, and 10 mL of anthrone reagent was rapidly added. The test tubes were capped and shaken thoroughly to ensure proper mixing of the reagent and filtrate. The capped tubes were then placed in a boiling water bath (approximately 100℃) for 20 min. to initiate the color reaction. Afterward, they were cooled in running tap water for 10 min. and vortexed to mix. The WSC content was analyzed using the Libra S70 Double Beam Spectrophotometer (Biochrom, Korea) at 620 nm wavelength. The spectrophotometer was adjusted to “0” using a blank, and then the WSC of standard and sample extractions were measured at 620 nm wavelength. The formula utilized to determine the Water-Soluble Carbohydrate (WSC) content is as follows: The percentage of WSC is calculated by multiplying the value of G (milligrams of glucose read from the graph) by the dilution factor (D), the extract volume (E, set at 200 mL), and a constant (0.1). This product is then multiplied by 100 and divided by the product of the sample weight (W, measured in milligrams) and the sample's laboratory dry matter percentage.

    The in vitro dry matter digestibility (IVDMD) was analyzed using the two-stage technique method detailed by Tilley and Terry (1963). Nylon filter bags were first soaked in acetone for 5 min and then dried in an air-forced drying oven at 105℃ for over 4 h. Ground samples weighing between 0.5 to 0.6 g were placed into the labeled nylon filter bags, which were then sealed tightly using a heat sealer. These sealed sample bags, along with 2 blank bags, were placed into incubation bottles (Ankom Technologies, Inc., Fairport, NY, USA) containing 1330 mL of buffer solution A and 266 mL of buffer solution B (in a 5:1 ratio, v/v). The incubation bottles were then placed in an incubator set at 39℃.

    Rumen fluid was collected from cows with cannulas. Holstein heifers before their morning feeding. The collected rumen fluid was pooled and filtered through four layers of cheesecloth into preheated thermos bottles. To each prepared incubation bottle, 400 mL of filtered rumen fluid was added. The bottles were then continuously filled with CO2 gas for 30 sec and tightly closed to achieve anaerobic conditions. The preparation of In vitro culture solution by combining rumen fluid and buffer solution at a ratio of 1:4. The incubation bottles were placed back into the incubator at 39℃ and continuously rotated for 48 h. After 48 h, the incubation bottles were removed, and the sample bags were washed until the water ran clear. The neutral detergent fiber (NDF) procedure was then performed. The buffer solution A contains the following components per liter: 10.0 g of KH2PO4, 0.5 g of MgSO4 · 7 H2O, 0.5 g of NaCl, 0.1 g of CaCl2 · 2H2O, and 0.5 g of reagent-grade urea. On the other hand, buffer solution B consists of 15.0 g of Na2CO3 and 1.0 gram of Na2S · 9H2O per liter.

    Total Digestible Nutrients (TDN) were calculated using the formula TDN% = 88.9 - (0.79 × ADF%). Relative Feed Value (RFV), which indicates feed quality, was derived from digestible dry matter (DDM) and dry matter intake (DMI) calculations based on ADF and NDF percentages.

    4. Statistical analysis

    All data were analyzed for variance by the General linear modal (GLM) procedure of SPSS statistical software (IBM SPSS Statistics 26 program SPSS Inc., Chicago, Illinois, USA). Mean treatment differences were obtained by Duncan’s multiple range tests. The significant differences were significant of 5%.

    Ⅲ. RESULTS AND DISCUSSION

    1. The agronomic characteristics and chemical composition of Italian ryegrass

    The agronomic characteristics and yields of Italian ryegrass at harvest are detailed in Table 1. The study recommended harvesting Italian ryegrass at the heading stage for optimal silage production using the round bale system, which correlates with the stage when the nutritional value is highest (Kim et al., 2001). The plant height of the Italian ryegrass was recorded at 97.40 cm. The yields were noted as 29,700 kg/ha for fresh matter and 7,446 kg/ha for dry matter.

    Studies have indicated that maintaining a moisture content between 65% and 75% is advisable for effective forage ensiling (McDonald et al., 1991). In this experiment, wilting was utilized to lower the moisture content at 25.07%, thereby improving silage attributes to achieve high-quality output. Wilting serves not only to effectively decrease moisture levels but also to enhance silage fermentation characteristics by inhibiting undesirable microorganism, preventing silo leakage loss, as suggested by Borreani (2018).

    2. The change of chemical composition of Italian ryegrass during ensiling

    The changes in dry matter (DM) and crude protein (CP) content of Italian ryegrass throughout ensiling are illustrated in Table 2. The DM content displayed a gradual change during the entire ensiling process, with more significant alterations occurring during the stages of silage fermentation and aerobic exposure (Ferraretto et al., 2018). The DM content stands as a crucial indicator in silage (Oladosu et al., 2016). According to Fan et al. (2022) as fermentation progressed, the DM content in the silage steadily declined. This decrease could be attributed to the utilization of WSC by fermentative microbes (Oladosu et al., 2016).

    The CP levels in Italian ryegrass silages, across all treatments, initially decreased within 1 to 2 days of ensiling. During the ensiling process, initial proteolytic changes occur. The extent of proteolysis during wilting hinges on the speed and magnitude of water loss. Kemble and Macpherson (1954) noted that over a 3 day wilting period, more than 20% of plant protein degraded into non-protein nitrogen (NPN). Two recognized stages are involved in the protein degradation process: Initially, peptide bond hydrolysis occurs, leading to the formation of free amino acids and peptides. Subsequently, amino acids undergo further degradation into various end products, including ammonia, organic acids, and amines. The initial phase is primarily driven by plant proteases released upon cellular damage, while microbial proteases largely dominate the subsequent phase (Wen et al., 2017).

    Throughout ensiling, CP content was consistently higher in all treatment groups compared to the control group, suggesting that the Lactiplantibacillus inoculant did not lead to nutrient depletion during the ensiling process (Guo et al., 2022). Notably, the T2 treatment, incorporating a combination of Lactiplantibacillus plantarum and Pediococcus pentosaceus, displayed even higher CP content at (105.53 g/kg DM) than T1 (87.95 g/kg DM), which included only Lactiplantibacillus plantarum. The CP content exhibited a consistent decline from day 1 to day 30. This decrease was observed as fermentation progressed, indicating that microorganisms consumed nutrients, leading to the gradual reduction in CP content in the silage (Dong et al., 2022).

    The Table 3 illustrates the significant impact of silage inoculants and ensiling duration on the changes in NDF, ADF, and hemicellulose during ensiling (p<0.05). On the 30th day, the NDF and ADF of Italian ryegrass silage treated with control were notably lower at 470.67 g/kg and 279.22 g/kg respectively, compared to other silages (p<0.05). This indicates degradation of the cell wall by plant enzymes, cellulolytic microorganisms, and acid hydrolysis. However, from day 30 to day 45, both NDF and ADF showed an increasing trend. Herrmann et al. (2015) reported that silage increases the relative proportion of fiber composition, which becomes more resistant to degradation over time. Similarly, Iptas and Acar (2006) observed a linear increase in NDF by 31 g/kg DM during ensiling. Santos et al. (2014) noted that both homo-fermentative and hetero-fermentative inoculants, as well as combined inoculants, increased NDF and ADF content. In agreement with these findings, Graybill et al. (1991) reported minimal differences in NDF and ADF content across various plant densities.

    This result aligns with Keady and Steen (1996), who found that inoculants has no significant effect or even increased NDF and ADF content. Similarly Santos et al. (2011) report escalated levels of NDF, ADF, and ADIN (Acid Detergent Insoluble Nitrogen) in signal grass silages across various regrowth intervals.

    In this experiment the hemicellulose content decreased during the ensiling from day 1 to day 20. This finding was consistent with the results of Iptas and Acar (2006), who observed a notable decrease in hemicellulose content during ensiling, indicating a similar trend. The hemicellulose content in the control treatment was significantly lower than in the other treatments, suggesting more efficient degradation of the cell wall into absorbable nutrients for animals (Wang & McAllister, 2002). Ning et al. (2017) found that hemicellulose activity progressively decreased, becoming inactive by day 14 post-ensiling. This result is consistent with Yahaya et al. (2002). Variability in cellulose degradation during ensiling might stem from its structural variations; some cellulose is protected by lignin, while other parts remain unaffected by lignin, dictating the rate of hydrolysis. After ensiling, hemicellulose contents in silage was slightly lower than that of the pre-ensiling material, likely due to acid hydrolysis of hemicelluloses at low pH value (McDonald et al., 1991;Buxton and O'Kiely, 2003).

    3. The change of IVDMD, RFV and TDN content of Italian ryegrass silage during ensiling

    The impact of ensiling duration and silage inoculant on IVDMD, RFV, and TDN content of Italian ryegrass silage is presented in Table 4. At present, the in vitro digestion technique is the sole accurate laboratory method for predicting in vivo digestibility. A strong correlation between in vivo and in vitro digestibility can be achieved if the procedure is well standardized (Mahyuddin, 2008). IVDMD initially increased, followed by a decrease and eventual stabilization during ensiling. In this experiment, the IVDMD of Italian ryegrass silages ware increased trend and 45 day control was significantly higher at 787.39 g/kg than other treatment. Gao et al. (2019) found that adding LAB and cellulase improved in vitro digestibility of corn stover silage. However, Liu et al. (2021) reported the no-additive group exhibited higher in vitro digestibility than the additive group. As reported by De Jesus Ferreira et al. (2013), the microbial inoculation had no impact on in vitro dry matter digestibility of elephant grass silage.

    RFV serves as an indicator of feed intake and digestibility, combining estimated intake of NDF and digestibility of ADF into one index. The relative feed value tends to decrease as ADF and NDF increase; higher levels of these traits are associated with lower relative feed values (Ashoori et al., 2021). RFV demonstrated an overall increase from the initial ensiling period to day 30. This effect could stem from enhanced substrate accessibility due to changes in the structural matrix of cell wall carbohydrates, potentially facilitating subsequent metabolism by aerobic organisms (He et al., 2000).

    TDN, which is directly linked to digestive energy and calculated based on ADF, showed an overall increasing trend during ensiling. By day 45, TDN in all silages treated with inoculants remained stable. Junior et al. (2020) showed that the percentages of evaluated nutrients in both control and inoculated silage samples remained constant throughout each stage. Additionally, TDN levels in Italian ryegrass silages increased compared to pre-ensiling Italian ryegrass. In this experiment, the inoculants had no significant effect on TDN. However, Li et al. (2023) reported variations in TDN and IVDMD among treatments throughout the ensiling period, indicating inconsistencies in the silages. Moreover, this treatment increased the nitrogen degradability in the rumen. According to the study by Weinberg et al. (2003), lactic acid bacteria (LAB) strains could migrate from silage to rumen fluid and persist there for at least 96 h. These LAB strains might engage with rumen microorganisms, potentially improving rumen functionality and enhancing animal performance. However, the precise cause remains unclear, necessitating further research in this area.

    4. Change of organic acid content and pH of Italian ryegrass silage during ensiling

    The pH is a traditional and crucial indicator for evaluating silage quality, with its decline primarily attributed to accumulation of organic acids, particularly lactic acid concentrations. Variances in lactic acid and acetic acid concentrations lead to differences in silage pH. Both the duration of ensiling and the use of silage additives significantly affected pH levels, which decreased and then stabilized. Silages treated with inoculants (T1, T2, T3, T4, and T5) exhibited lower pH levels compared to the control group (p<0.05), with stabilization occurring after the 10th day of ensiling. All treatments recorded pH levels below 4.2, indicating effective preservation of the silage (Wen et al., 2017). Liu et al. (2021) similarly reported that inoculated silages had significantly lower pH levels. A study by Shao et al. (2002) on the fermentation dynamics of Italian ryegrass silage over 14 days revealed substantial reductions in pH, especially within the first 5 days of ensiling. The speed of pH decline is crucial, as a rapid decrease reduces the risk of initial growth of undesirable microorganisms (Makoni et al., 1997). The inoculant containing Lactiplantibacillus plantarum achieved a pH of 4.0 within four days, demonstrating that even a single bacterial strain can produce silage of high quality. It's well-documented that a substantial population of LAB is essential for a rapid, strong fermentation, leading to a rapid pH drop and high accumulation of lactic acid. These factors are critical for ensuring superior crop quality during ensiling (Ennahar et al., 2003). Inoculation has proven to be an effective method for enhancing silage quality by facilitating the swift and efficient utilization of a crop's WSC, which leads to intensified lactic acid production, a rapid pH decrease, and the suppression of detrimental microbes (Ennahar et al., 2003).

    Lactic acid (LA) has the highest acidity level among organic acids, such as acetic acid, propionic acid, and butyric acid (Moon, 1983). Throughout the fermentation period, an observable increase in LA content was noted with the control treatment showing significantly lower levels compared to the other group. Due to the strong acid tolerance and homofermentative characteristics of LAB, the WSC content was converted into LA content in LAB-treated silages significantly surpassed that of the control group, likely due to the higher prevalence of LAB in these treatments. The performance of LAB dictates the buildup of LA and the rate at which pH decreases in the early stages of ensiling (Davies et al., 1998). Italian ryegrass silage treated with T1, T2, T3, T4, and T5 showed a rapid increase in LA content within the first 10 days. By day 45, T3 treatment exhibited the highest LA content at 120.38 g/kg DM. The rise in LA content was consistent during the ensiling process, with a faster increase observed in the initial 5 days, followed by gradual stabilization, which align with the criteria for favorable fermentation.

    Acetic acid (AA) is the second-highest concentrated acid found in silages, which aligns with findings of Kung Jr et al. (2018). In contrast, the acetic acid content in the control treatment demonstrated a gradual increase, ultimately reaching the highest level among all treatments. Italian ryegrass silage treated with control, T1, and T4 showed a rapid increase in AA content by the 4th day, reaching levels of 14.61, 6.14, and 5.98 g/kg DM, respectively. By the 45th day, the control treatment recorded the highest AA content at 23.54 g/kg DM, followed by T3 at 16.07 g/kg DM. The higher AA content in T3 can be attributed to the heterofermentative activity of Lactiplantibacillus buchneri present in this treatment. Driehuis et al. (2001) reported that the introduction of Lactiplantibacillus buchneri through inoculation leads to an increase in acetic acid concentration within silage. Additionally, combining Lactiplantibacillus buchneri with homofermentative LAB is preferred, as it this approach not only accelerates the initial lactic acid fermentation but also minimizes protein degradation and reduces dry matter loss. The decrease in pH within the first 3 days might be explained by the substantial production of AA, despite its accounting for a smaller proportion than LA. Throughout this experiment, LA content in all silages surpassed AA content, indicating the dominance of lactic acid fermentation.

    The propionic acid (PA) is frequently used to prevent heat generation in silage and enhance aerobic stability when exposed to open air due to its acidic nature and antimicrobial properties (He et al., 2020). It has been found that treating with propionic acid improves the fermentation quality of whole-plant barley silage by reducing pH, decreasing butyric acid content, and increasing lactic acid content (Kung Jr and Ranjit, 2001). Regarding PA content, control and T4 treatments exhibited an increase over the ensiling period, whereas no PA content was detected in the T2 and T5 groups. By the 45th day, the highest PA content was observed in the T4 treatment at 5.51 g/kg DM.

    5. The change of WSC and NH3-N/TN of Italian ryegrass silage during ensiling

    During fermentation, the WSC content in all Italian ryegrass silages gradually diminished, as it was consumed by lactic acid bacteria and other microbes. This decline was particularly rapid during the initial 4 days, after which it nearly stabilized. The initial stage of ensiling is driven by the enzymatic activity within intact plant cells, often referred to as residual respiration. During this phase, these cells utilize the oxygen present within the silage and metabolize carbohydrates such as glucose and fructose (Shao et al., 2005). The consumption of carbohydrates in the early stages can be detrimental to subsequent anaerobic lactic acid fermentation, which is crucial for effective silage preservation. During this initial phase, aerobic microorganisms proliferate until oxygen is fully depleted or until sufficient acidification halts their metabolic activity. This utilization of carbohydrates was substantial, primarily due to a large population of lactic acid bacteria, which produced significant amounts of lactic acid, lowering the pH and hindering the growth of undesirable bacteria (Heron et al., 1993). By the 45 day, the soluble carbohydrate content had dropped significantly to approximately 3.5 and 2.4%, respectively. Fan et al. (2022) reported the gradual decrease in WSC content in silages occurred due to the utilization of nutrients by microorganisms.

    Ammonia nitrogen is a crucial indicator for evaluating the fermentation quality of silage. A higher NH3-N to total nitrogen (TN) ratio indicates a greater breakdown of amino acids and proteins (Denek et al., 2017). The production of ammonia nitrogen is primarily associated with the metabolic activity of Clostridium bacteria, which ferment amino acids to ammonia. Elevated level of NH3-N/TN typically signify inadequate fermentation (McDonald et al., 1991). Throughout the ensiling process, the NH3-N/TN content in Italian ryegrass silages steadily increased, with a notably higher rate of increase observed by the third day. The control silage exhibited a significantly greater rise in NH3-N/TN compared to the inoculated silages. After 45 days, the NH3-N/TN content in Italian ryegrass treated with T4 (3.09%) was considerably lower than in the control (6.47%). In this experiment the NH3-N/TN concentration remained below 10%. According to Costa et al. (2016), effectively fermented silage is expected to have NH3-N contents below 10% of total nitrogen. This validates that silage additives positively impact the rapid decrease in pH values, leading to a favorable reduction in protein degradation within the silo (Nkosi et al., 2010). At the later stages of ensiling, there was a noticeable prevalence of Clostridium, primarily proteolytic clostridia rather than saccharolytic clostridia (Yin et al., 2023). Inoculating with T4 significantly suppressed protein degradation by Clostridium. The reduction in peptide degradation may be linked to the inactivation of peptidase activity, which could result from the lower pH or the binding of peptides with phenol during the initial stages of ensiling. This interaction between peptides and phenol might make the peptides less accessible to peptidases in the later stages of ensiling (Guo et al., 2007). Similar to peptides, free amino acids are the products of proteolysis initiated by plant proteases in the early stages of ensiling. The concentrations of free amino acid nitrogen (N) are closely associated with the degree of proteolysis (Guo et al., 2011). Certain strain of clostridia possess diverse metabolic capabilities, including fermenting sugars into butyric acid, converting lactic acid into butyric acid, and exhibiting significant proteolytic activity (Kung Jr et al., 2018). In clostridial silages, in addition to elevated levels of butyric acid and lower-thanusual concentrations of lactic acid, there's often an increase in pH and higher concentrations of acetic acid, NH3-N, and soluble protein compared to standard silages (Mills and Kung Jr, 2002).

    Ammonia is generated as a byproduct of further amino acid breakdown through microbial proteolysis rather than plantmediated proteolysis (Guo et al., 2007). As ensiling processes, microbial proteolysis tends to become more prevalent due to increased microbial activity (Dong et al., 2019).

    Ⅳ. CONCLUSIONS

    Based on the experimental findings from the Italian ryegrass silage study, it can be concluded that using various inoculants during the ensiling significantly improves both the preservation quality and nutritional profile of the silage. The higher CP content in the treatment groups compared to the control indicates that inoculants help preserve protein level in the silage. The consistent decrease in NDF, ADF, and hemicellulose suggests enhanced fiber digestibility. This is supported by the initial increase in IVDMD. The overall rise in RFV and the trend of increasing TDN during ensiling highlight the enhanced nutritional value of the treated silage.

    The significant reduction and stabilization of pH levels below 4.2 in the inoculant-treated silages demonstrate effective fermentation and preservation. Increased LA content and the favorable LA/AA ratio in the T2 group further confirm the improved fermentation quality associated with inoculant use. The observed decrease in WSC and the increase in NH3-N/TN suggest that active microbial fermentation occurs, particularly in the early stages of ensiling.

    The studied inoculants, especially Lactiplantibacillus plantarum and Pediococcus pentosaceus, effectively enhanced lactic acid fermentation and improved silage preservation. Lactiplantibacillus buchneri and Enterococcus faecium also showed promising results in preserving nutrients. Overall, the findings suggest that using specific inoculants boosts fermentation, leading to better preservation and improved nutritional quality of Italian ryegrass silage.

    Ⅴ. ACKNOWLEDGEMENTS

    This research was financially supported by the Cooperative Research Program for Agriculture Science & Technology Development (Project No. RS-2021-RD010124), Rural Development Administration, Republic of Korea.

    Figure

    KGFS-44-4-226_F1.gif

    Effect of inoculants on the dynamic change of pH of Italian ryegrass silage. T1=Lactiplantibacillus plantarum at the rate of 3×105 cfu/50 kg; T2=Lactiplantibacillus plantarum and Pediococcus pentosaceus at the rate of 2×104 cfu/50 kg; T3=Lactiplantibacillus plantarum and Pediococcus pentosaceus and Lactiplantibacillus buchneri at the rate of 2.5×105 cfu/ 50kg; T4=Lactiplantibacillus plantarum and Lactiplantibacillus acidophilus and Lactiplantibacillus bulgaricus at the rate of 4.25×103 cfu/ 50kg; T5=Lactiplantibacillus plantarum and Pediococcus pentosaceus and Enterococcus faecium at the rate of 5×105 cfu/ 50kg; D: ensiling days, I: Inoculants.

    KGFS-44-4-226_F2.gif

    Effect of inoculants on the dynamic change of WSC of Italian ryegrass silage. T1=Lactiplantibacillus plantarum at the rate of 3×105 cfu/50 kg; T2=Lactiplantibacillus plantarum and Pediococcus pentosaceus at the rate of 2×104 cfu/50 kg; T3=Lactiplantibacillus plantarum and Pediococcus pentosaceus and Lactiplantibacillus buchneri at the rate of 2.5×105 cfu/ 50kg; T4=Lactiplantibacillus plantarum and Lactiplantibacillus acidophilus and Lactiplantibacillus bulgaricus at the rate of 4.25×103 cfu/ 50kg; T5=Lactiplantibacillus plantarum and Pediococcus pentosaceus and Enterococcus faecium at the rate of 5×105 cfu/ 50kg; D: ensiling days, I: Inoculants.

    KGFS-44-4-226_F3.gif

    Effect of inoculants on the NH3-N/TN of Italian ryegrass silage during ensiling. T1=Lactiplantibacillus plantarum at the rate of 3×105 cfu/50 kg; T2=Lactiplantibacillus plantarum and Pediococcus pentosaceus at the rate of 2×104 cfu/50 kg; T3=Lactiplantibacillus plantarum and Pediococcus pentosaceus and Lactiplantibacillus buchneri at the rate of 2.5×105 cfu/ 50kg; T4=Lactiplantibacillus plantarum and Lactiplantibacillus acidophilus and Lactiplantibacillus bulgaricus at the rate of 4.25×103 cfu/ 50kg; T5=Lactiplantibacillus plantarum and Pediococcus pentosaceus and Enterococcus faecium at the rate of 5×105 cfu/ 50kg; D: ensiling days, I: Inoculants.

    Table

    Agronomic characteristics and yield of Italian ryegrass

    Effect of different inoculants on the dynamic change of DM, CP content of Italian ryegrass silage

    DM=dry matter; CP=crude protein. a – f Mean within rows with unlike superscripts differ (p<0.05); A- C Means within columns with unlike superscripts differ (p<0.05); T1=Lactobacillus plantarum at the rate of 3×105 cfu/50 kg; T2=Lactiplantibacillus plantarum and Pediococcus pentosaceus at the rate of 2×104 cfu/50 kg; T3=Lactiplantibacillus plantarum and Pediococcus pentosaceus and Lactiplantibacillus buchneri at the rate of 2.5×105 cfu/ 50kg; T4=Lactiplantibacillus plantarum and Lactiplantibacillus acidophilus and Lactiplantibacillus bulgaricus at the rate of 4.25×103 cfu/ 50kg; T5=Lactiplantibacillus plantarum and Pediococcus pentosaceus and Enterococcus faecium at the rate of 5×105 cfu/ 50kg.

    Effect of different inoculants on the dynamic change of NDF, ADF and hemicellulose content of Italian ryegrass silage

    DM=dry matter; NDF= neutral detergent fiber; ADF=acid detergent fiber. a – f Mean within rows with unlike superscripts differ (p<0.05); AF Means within columns with unlike superscripts differ (p<0.05). T1=Lactiplantibacillus plantarum at the rate of 3×105 cfu/50 kg; T2=Lactiplantibacillus plantarum and Pediococcus pentosaceus at the rate of 2×104 cfu/50 kg; T3=Lactiplantibacillus plantarum and Pediococcus pentosaceus and Lactiplantibacillus buchneri at the rate of 2.5×105 cfu/ 50kg; T4=Lactiplantibacillus plantarum and Lactiplantibacillus acidophilus and Lactiplantibacillus bulgaricus at the rate of 4.25×103 cfu/ 50kg; T5=Lactiplantibacillus plantarum and Pediococcus pentosaceus and Enterococcus faecium at the rate of 5×105 cfu/ 50kg.

    Effect of different inoculants on the dynamic change of IVDMD, RFV and TDN content of Italian ryegrass silage

    DM=dry matter; IVDMD=in vitro dry matter digestibility; RFV= relative feed value; TDN=total digestible nutrient. a – d Mean within rows with unlike superscripts differ (p<0.05); A- E Means within columns with unlike superscripts differ (p<0.05). T1=Lactiplantibacillus plantarum at the rate of 3×105 cfu/50 kg; T2=Lactiplantibacillus plantarum and Pediococcus pentosaceus at the rate of 2×104 cfu/50 kg; T3=Lactiplantibacillus plantarum and Pediococcus pentosaceus and Lactiplantibacillus buchneri at the rate of 2.5×105 cfu/ 50kg; T4=Lactiplantibacillus plantarum and Lactiplantibacillus acidophilus and Lactiplantibacillus bulgaricus at the rate of 4.25×103 cfu/ 50kg; T5=Lactiplantibacillus plantarum and Pediococcus pentosaceus and Enterococcus faecium at the rate of 5×105 cfu/ 50kg.

    Effect of different inoculants on the dynamic change of organic acid content of Italian ryegrass silage

    DM=dry matter; LA=lactic acid; AA=acetic acid; PA=propionic acid. a – d Mean within rows with unlike superscripts differ (p<0.05); A- G Means within columns with unlike superscripts differ (p<0.05); T1=Lactiplantibacillus plantarum at the rate of 3×105 cfu/50 kg; T2=Lactiplantibacillus plantarum and Pediococcus pentosaceus at the rate of 2×104 cfu/50 kg; T3=Lactiplantibacillus plantarum and Pediococcus pentosaceus and Lactiplantibacillus buchneri at the rate of 2.5×105 cfu/ 50kg; T4=Lactiplantibacillus plantarum and Lactiplantibacillus acidophilus and Lactiplantibacillus bulgaricus at the rate of 4.25×103 cfu/ 50kg; T5=Lactiplantibacillus plantarum and Pediococcus pentosaceus and Enterococcus faecium at the rate of 5×105 cfu/ 50kg.

    Reference

    1. Ashoori, N., Abdi, M., Golzardi, F., Ajalli, J. and Ilkaee, M.N. 2021. Forage potential of sorghum-clover intercropping systems in semi-arid conditions. Bragantia. 80:e1421.
    2. Bai, J., Ding, Z., Ke, W., Xu, D., Wang, M., Huang, W., Zhang, Y., Liu, F. and Guo, X. 2021. Different lactic acid bacteria and their combinations regulated the fermentation process of ensiled alfalfa: Ensiling characteristics, dynamics of bacterial community and their functional shifts. Microbial Biotechnology. 14:1171-1182.
    3. Borreani, G., Tabacco, E., Schmidt, R., Holmes, B. and Muck, R.A. 2018. Silage review: Factors affecting dry matter and quality losses in silages. Journal of Dairy Science. 101:3952-3979.
    4. Broderick, G. and Kang, J. 1980. Automated simultaneous determination of ammonia and total amino acids in ruminal fluid and in vitro media. Journal of Dairy Science. 63:64-75.
    5. Buxton, D.R. and O'Kiely, P. 2003. Preharvest plant factors affecting ensiling. Silage Science and Technology. 42:199-250.
    6. Choi, G.J., Ji, H.C., Kim, K.Y., Park, H.S., Seo, S., Lee, K.W. and Lee, S.H. 2011. Growth characteristics and productivity of cold-tolerant “Kowinearly” Italian ryegrass in the northern part of South Korea. African Journal of Biotechnology. 10:2676-2682.
    7. Costa, R.F., Pires, D.A.A., Moura, M.M.A., Sales, E.C.J.D., Rodrigues, J.A.S. and Rigueira, J.P.S. 2016. Agronomic characteristics of sorghum genotypes and nutritional values of silage. Acta Scientiarum. Animal Sciences. 38:127-133.
    8. Davies, D., Merry, R., Williams, A., Bakewell, E.L., Leemans, D. and Tweed, J. 1998. Proteolysis during ensilage of forages varying in soluble sugar content. Journal of Dairy Science. 81:444-453.
    9. De Jesus Ferreira, D., de Paula Lana, R., de Moura Zanine, A., Santos, E.M., Veloso, C.M. and Ribeiro, G.A. 2013. Silage fermentation and chemical composition of elephant grass inoculated with rumen strains of Streptococcus bovis. Animal Feed Science and Technology. 183:22-28.
    10. Denek, N., Aydin, S.S. and Can, A. 2017. The effects of dried pistachio (Pistachio vera L.) by-product addition on corn silage fermentation and in vitro methane production. Journal of Applied Animal Research. 45:185-189.
    11. Dong, D., Xu, G., Dai, T., Zong, C., Yin, X., Bao, Y. and Shao, T. 2022. Effect of molasses on fermentation quality of wheat straw ensiled with perennial ryegrass. Animal Production Acience. 62:1471-1479.
    12. Dong, Z., Chen, L., Li, J., Yuan, X. and Shao, T. 2019. Characterization of nitrogen transformation dynamics in alfalfa and red clover and their mixture silages. Grassland Science. 65:109-115.
    13. Driehuis, F., Oude Elferink, S. and Van Wikselaar, P. 2001. Fermentation characteristics and aerobic stability of grass silage inoculated with Lactobacillus buchneri, with or without homofermentative lactic acid bacteria. Grass and Forage Science. 56:330-243.
    14. Dumas, J. 1831. Procédés de l’analyse organique. Ann. Chim. Phys. 47:198-205.
    15. Ennahar, S., Cai, Y. and Fujita, Y. 2003. Phylogenetic diversity of lactic acid bacteria associated with paddy rice silage as determined by 16S ribosomal DNA analysis. Applied and Environmental Microbiology. 69:444-451.
    16. Fan, X., Xie, Z., Cheng, Q., Li, M., Long, J., Lei, Y., Jia, Y., Chen, Y., Chen, C. and Wang, Z. 2022. Fermentation quality, bacterial community, and predicted functional profiles in silage prepared with alfalfa, perennial ryegrass and their mixture in the karst region. Frontiers in Microbiology. 13:1062515.
    17. Ferraretto, L., Silva Filho, W., Fernandes, T., Kim, D. and Sultana, H. 2018. Effect of ensiling time on fermentation profile and ruminal in vitro starch digestibility in rehydrated corn with or without varied concentrations of wet brewers grains. Journal of Dairy Science. 101:4643-4649.
    18. Gao, J.L., Wang, P., Zhou, C.H., Li, P., Tang, H.Y., Zhang, J.B. and Cai, Y. 2019. Chemical composition and in vitro digestibility of corn stover during field exposure and the fermentation characteristics of silage prepared with microbial additives. Asian-Australasian Journal of Animal Sciences. 32:1854.
    19. Gonda, H., Nikodinoska, I., Le Cocq, K. and Moran, C.A. 2023. Efficacy of six lactic acid bacteria strains as silage inoculants in forages with different dry matter and water‐soluble carbohydrate content. Grass and Forage Science. 78:636-647.
    20. Guo, X., Cheng, W., Zhang, Y., Yang, F. and Zhou, H. 2011. Contribution of endopeptidases to the formation of nonprotein nitrogen during ensiling of alfalfa. Animal Feed Science and Technology. 168:42-50.
    21. Guo, X., Guo, W., Yang, M., Sun, Y., Wang, Y., Yan, Y. and Zhu, B. 2022. Effect of Bacillus additives on fermentation quality and bacterial community during the ensiling process of whole-plant corn silage. Processes. 10:978.
    22. Guo, X., Zhou, H., Yu, Z. and Zhang, H. 2007. Changes in the distribution of nitrogen and plant enzymatic activity during ensilage of lucerne treated with different additives. Grass and Forage Science. 62:35-43.
    23. He, L., Zhou, W., Xing, Y., Pian, R., Chen, X. and Zhang, Q. 2020. Improving the quality of rice straw silage with Moringa oleifera leaves and propionic acid: Fermentation, nutrition, aerobic stability and microbial communities. Bioresource Technology. 299:122579.
    24. He, X., Agnihotri, G. and Liu, H.W. 2000. Novel enzymatic mechanisms in carbohydrate metabolism. Chemical Reviews. 100:4615-4662.
    25. Heron, S.J., Wilkinson, J. and Duffus, C.M. 1993. Enterobacteria associated with grass and silages. Journal of Applied Microbiology. 75:13-17.
    26. Herrmann, C., Idler, C. and Heiermann, M. 2015. Improving aerobic stability and biogas production of maize silage using silage additives. Bioresource Technology. 197:393-403.
    27. Iptas, S. and Acar, A. 2006. Effects of hybrid and row spacing on maize forage yield and quality. Plant Soil and Environment. 52:515.
    28. Jia, T., Wang, B., Yu, Z. and Wu, Z. 2021. The effects of stage of maturity and lactic acid bacteria inoculants on the ensiling characteristics, aerobic stability and in vitro digestibility of wholecrop oat silages. Grassland Science. 67:55-62.
    29. Keady, T. and Steen, R. 1996. Effects of applying a bacterial inoculant to silage immediately before feeding on silage intake, digestibility, degradability and rumen volatile fatty acid concentrations in growing beef cattle. Grass and Forage Science. 51:155-162.
    30. Kemble, A. and Macpherson, H.T. 1954. Liberation of amino acids in perennial rye grass during wilting. Biochemical Journal. 58:46.
    31. Kim, J., Chung, E., Seo, S., Ham, J., Kang, W. and Kim, D. 2001. Effects of maturity at harvest and wilting days on quality of round baled rye silage. Asian-Australasian Journal of Animal Sciences. 14:1233-1237.
    32. Kung Jr, L. and Ranjit, N. 2001. The effect of Lactobacillus buchneri and other additives on the fermentation and aerobic stability of barley silage. Journal of Dairy Science. 84:1149-1155.
    33. Kung Jr, L., Shaver, R., Grant, R. and Schmidt, R. 2018. Silage review: Interpretation of chemical, microbial, and organoleptic components of silages. Journal of Dairy Science. 101:4020-4033.
    34. Kung Jr, L., Stokes, M.R. and Lin, C. 2003. Silage additives. Silage Science and Technology. 42:305-360.
    35. Kung, L. 2018. Silage fermentation and additives. Latin American Archives of Animal Production. 26.
    36. Li, Y.F., Jeong, E.C., Wang, L.L., Kim, H.J., Ahmadi, F. and Kim, J.G. 2023. Effects of sodium diacetate and microbial inoculants on fermentation of forage rye. Journal of Animal Science and Technology. 65:96.
    37. Li, Y. and Nishino, N. 2011. Monitoring the bacterial community of maize silage stored in a bunker silo inoculated with Enterococcus faecium, Lactobacillus plantarum and Lactobacillus buchneri. Journal of Applied Microbiology. 110:1561-1570.
    38. Li, Y., Da Silva, E., Li, J. and Kung Jr, L. 2022. Effect of homo-fermentative lactic acid bacteria inoculants on fermentation characteristics and bacterial and fungal communities in alfalfa silage. Fermentation. 8:621.
    39. Lindgren, S.E., Axelsson, L.T. and McFeeters, R.F. 1990. Anaerobic L-lactate degradation by Lactobacillus plantarum. FEMS Microbiology Letters. 66:209-213.
    40. Liu, C., Zhao, G.Q., Wei, S.N., Kim, H.J., Li, Y.F. and Kim, J.G. 2021. Changes in fermentation pattern and quality of Italian ryegrass (Lolium multiflorum Lam.) silage by wilting and inoculant treatments. Animal Bioscience. 34:48.
    41. Lobo, C.B., Tomás, M.S.J., Viruel, E., Ferrero, M.A. and Lucca, M.E. 2019. Development of low-cost formulations of plant growth-promoting bacteria to be used as inoculants in beneficial agricultural technologies. Microbiological Research. 219:12-25.
    42. Mahyuddin, P. 2008. Relationship between chemical component and in vitro digestibility of tropical grasses. Hayati Journal of Biosciences. 15:85-89.
    43. Makoni, N., Broderick, G. and Muck, R. 1997. Effect of modified atmospheres on proteolysis and fermentation of ensiled alfalfa. Journal of Dairy Science. 80:912-920.
    44. McDonald, P., Henderson, A. and Heron, S.J.E. 1991. The biochemistry of silage. Chalcombe Publications.
    45. Mills, J. and Kung Jr, L. 2002. The effect of delayed ensiling and application of a propionic acid-based additive on the fermentation of barley silage. Journal of Dairy Science. 85:1969-1975.
    46. Moon, N.J. 1983. Inhibition of the growth of acid tolerant yeasts by acetate, lactate and propionate and their synergistic mixtures. Journal of Applied Microbiology. 55:453-460.
    47. Nilsson, R., Tóth, L. and Rydin, C. 1956. Studies on fermentation processes in silage: The role of temperature. Archiv für Mikrobiologie. 23:366-375.
    48. Ning, T., Wang, H., Zheng, M., Niu, D., Zuo, S. and Xu, C. 2017. Effects of microbial enzymes on starch and hemicellulose degradation in total mixed ration silages. Asian-Australasian Journal of Animal Sciences. 30:171.
    49. Nkosi, B., Meeske, R., Van der Merwe, H. and Groenewald, I. 2010. Effects of homofermentative and heterofermentative bacterial silage inoculants on potato hash silage fermentation and digestibility in rams. Animal Feed Science and Technology. 157:195-200.
    50. Oladosu, Y., Rafii, M.Y., Abdullah, N., Magaji, U., Hussin, G., Ramli, A. and Miah, G. 2016. Fermentation quality and additives: A case of rice straw silage. BioMed Research International. 2016:7985167.
    51. Oude Elferink, S.J., Krooneman, J., Gottschal, J.C., Spoelstra, S.F., Faber, F. and Driehuis, F. 2001. Anaerobic conversion of lactic acid to acetic acid and 1, 2-propanediol by Lactobacillus buchneri. Applied and Environmental Microbiology. 67:125-132.
    52. Parvin, S. and Nishino, N. 2010. Succession of lactic acid bacteria in wilted rhodesgrass silage assessed by plate culture and denaturing gradient gel electrophoresis. Grassland Science. 56:51-55.
    53. Rohweder, D., Barnes, R. and Jorgensen, N. 1978. Proposed hay grading standards based on laboratory analyses for evaluating quality. Journal of Animal Science. 47:747-759.
    54. Santos, E.M., Pereira, O.G., Garcia, R., Ferreira, C.L.D.L.F., Oliveira, J.S.D., Silva, T.C.D. and Rosa, L.O. 2011. Microbial populations, fermentative profile and chemical composition of signalgrass silages at different regrowth ages. Revista Brasileira de Zootecnia. 40:747-755.
    55. Santos, E., Pereira, O., Garcia, R., Ferreira, C., Oliveira, J. and Silva, T. 2014. Effect of regrowth interval and a microbial inoculant on the fermentation profile and dry matter recovery of guinea grass silages. Journal of Dairy Science. 97:4423-4432.
    56. Shao, T., Ohba, N., Shimojo, M. and Masuda, Y. 2002. Dynamics of early fermentation of Italian ryegrass (Lolium multiflorum Lam.) silage. Asian-Australasian Journal of Animal Sciences. 15:1606-1610.
    57. Shao, T., Zhang, Z., Shimojo, M., Wang, T. and Masuda, Y. 2005. Comparison of fermentation characteristics of Italian ryegrass (Lolium multiflorum Lam.) and guineagrass (Panicum maximum Jacq.) during the early stage of ensiling. Asian-Australasian Journal of Animal Sciences. 18:1727-1734.
    58. Tilley, J. and Terry, D.R. 1963. A two-stage technique for the in vitro digestion of forage crops. Grass and Forage Science. 18:104-111.
    59. Van Soest, P.V., Robertson, J.B. and Lewis, B.A. 1991. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. Journal of Dairy Science. 74:3583-3597.
    60. Wang, Y. and McAllister, T. 2002. Rumen microbes, enzymes and feed digestion-a review. Asian-Australasian Journal of Animal Sciences. 15:1659-1676.
    61. Weinberg, Z., Muck, R. and Weimer, P. 2003. The survival of silage inoculant lactic acid bacteria in rumen fluid. Journal of Applied Microbiology. 94:1066-1071.
    62. Wen, A., Yuan, X., Wang, J., Desta, S.T. and Shao, T. 2017. Effects of four short-chain fatty acids or salts on dynamics of fermentation and microbial characteristics of alfalfa silage. Animal Feed Science and Technology. 223:141-148.
    63. Yahaya, M., Kawai, M., Takahashi, J. and Matsuoka, S. 2002. The effect of different moisture contents at ensiling on silo degradation and digestibility of structural carbohydrates of orchardgrass. Animal Feed Science and Technology. 101:127-133.
    64. Yemm, E. and Willis, A. 1954. The estimation of carbohydrates in plant extracts by anthrone. Biochemical Journal. 57:508.
    65. Yin, X., Zhao, J., Wang, S., Dong, Z., Li, J. and Shao, T. 2023. The effects of epiphytic microbiota and chemical composition of Italian ryegrass harvested at different growth stages on silage fermentation. Journal of the Science of Food and Agriculture. 103:1385-1393.
    66. You, S., Du, S., Ge, G., Wan, T. and Jia, Y. 2021. Selection of lactic acid bacteria from native grass silage and its effects as inoculant on silage fermentation. Agronomy Journal. 113:3169-3177.
    67. Yu, Y.S., Li, Y.F., Panyavong, X., Wu, L.Z., Hwang, J.U., Wang, L.L. and Kim, J.G., 2024. Comparison of treatment effect of domestically distributed major silage inoculant. Journal of The Korean Society of Grassland and Forage Science. 44:50-57.
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