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Article

The Influence of Silage Additives Supplementation on Chemical Composition, Aerobic Stability, and In Vitro Digestibility in Silage Mixed with Pennisetum giganteum and Rice Straw

College of Coastal Agricultural Sciences, Guangdong Ocean University, Zhanjiang 524088, China
*
Author to whom correspondence should be addressed.
Agriculture 2024, 14(11), 1953; https://doi.org/10.3390/agriculture14111953
Submission received: 25 September 2024 / Revised: 25 October 2024 / Accepted: 30 October 2024 / Published: 31 October 2024
(This article belongs to the Section Farm Animal Production)

Abstract

:
The purpose of the current research was to evaluate the influence of lactic acid bacteria and cellulase supplementation on the chemical composition, fermentation parameters, aerobic stability, microbial count, and in vitro nutrients digestibility of silage prepared with Pennisetum giganteum and rice straw. This study consisted of four treatments: a control group with no additive supplementation (CON), a lactic acid bacteria supplementation group (LAB), a cellulase supplementation group (CEL), and a combined supplementation group (LAC). After ensiling for 60 d, the chemical composition, fermentation parameters, microbial count, and aerobic stability were determined. Additionally, ruminal fermentation characteristics were evaluated by an in vitro incubation technique. Compared with CON silage, the quality of LAB and CEL silages was enhanced to a certain degree. Combined supplementation with lactic acid bacteria and cellulase in mixed silage of Pennisetum giganteum and rice straw noticeably increased (p < 0.05) the dry matter, crude protein, and lactic acid contents, whereas it reduced (p < 0.05) the pH and ammonia nitrogen/total nitrogen as well as the neutral detergent fiber and acid detergent fiber concentrations. The lactic acid bacteria count in LAC silage was higher (p < 0.05) than that of CON silage, whereas an opposite trend of yeast, aerobic bacteria, and mold was observed between the two groups. The aerobic stability time, in vitro crude protein, and neutral detergent fiber digestibility in LAC silage were significantly increased (p < 0.05) compared with those in CON silage. Moreover, the in vitro ruminal ammonia nitrogen content was reduced (p < 0.05), and the microbial protein and propionic acid concentrations were increased (p < 0.05) in silage after combined inoculation with additives. Taken together, the quality of Pennisetum giganteum and rice straw mixed silage can be improved by inoculation with lactic acid bacteria and cellulase, and combined supplementation shows the greatest improvement in silage quality.

1. Introduction

In ruminant production, an adequate supply of roughage of high quality plays a critical role in improving ruminal function and productivity [1]. Alfalfa is a frequently used hay in the diet of ruminants. In addition, as an effective method for improving the quality of roughage and expanding feed sources, silage fodder is commonly utilized in ruminant feed production [2]. Corn is an important plant source for ensiling. However, alfalfa and corn have high requirements for water. The yield of alfalfa and corn is poor in some regions because of poor soils as well as limited land and water resources [3]. On the other hand, the nutrient contents, such as crude protein (CP), and the nutrients degradation rate in the rumen of maize ensiling are lower than those of grass silage [4]. In some parts of the world, the shortage in roughage of high quality seriously restricts the productivity of the ruminant industry. The utilization of non-conventional feed resources in ruminant farming, which are characterized by high yield and nutrient contents, has attracted more and more attention.
Plants that have the ability to grow in a severe environment of water shortages, saline–alkali soils, and high temperatures can be used as roughage for ruminants in some areas [5,6]. The Pennisetum giganteum (P. giganteum), a C4 crop with a highly efficient photosynthetic pathway belonging to the genus Pennisetum, can grow in regions with a harsh environment [7]. Accordingly, the yield of P. giganteum is high, and it has the characteristics of good palatability and nutritional value [8]. Based on our previous investigation, the P. giganteum harvested at 2 m plant height (elongation stage) has a yield as high as 190 t/hectare (fresh weight) and 26 t/hectare [dry matter (DM)], respectively [9]. Also, the concentrations of nitrate and oxalic acid, which can affect the nutrients digestion of animals, are lower in the P. giganteum compared with some crops, including corn straw and sudangrass [10]. In the 2 m plant height of P. giganteum, the CP and water soluble carbohydrate (WSC) concentrations of the whole plant are approximately 13.8% and 7.2% (DM basis), respectively [9]. Additionally, P. giganteum is rich in a variety of amino acids, and the content of total amino acids is up to 7.1% [10]. Because of all these characteristics, P. giganteum can be utilized as a roughage resource in ruminant feed production. In the diet of goats [11] and dairy cows [12], the partial substitution of corn silage and concentrates by forage P. giganteum does not show adverse effects on immunity and production performance of animals but improves economic benefits.
Our research found that the appropriate growth stage of P. giganteum for making silage was 2 to 2.5 m plant height (approximately the elongation stage) [9]. Although P. giganteum can be used for ensiling, the quality is low due to the high content of moisture (approximately 88%) that can dilute the WSC content and inhibit lactobacillus fermentation [13]. Moisture content in the raw material is an essential factor affecting the fermentation process of silage [13]. In agricultural countries, the yield of crop straw is abundantly available, and the inappropriate management of crop straw will contaminate soil and cause air pollution [14]. Crop straw is rich in DM [14]; thus, we selected P. giganteum and rice straw as the raw materials for ensiling in this experiment. Moreover, the use of additives, including lactic acid bacteria and cellulase, is an effective method to improve silage quality [15,16]. On the other hand, a key index of nutrient value evaluation in roughage is ruminal utilization, and the in vitro ruminal incubation technique is generally used to assess the nutrient value of feedstuff [17]. At present, there is no sufficient data related to the influence of ensiling additives on the quality of P. giganteum and rice straw mixed silage. Therefore, this experiment was performed to assess the influence of lactic acid bacteria and cellulase supplementation on the chemical composition, fermentation parameters, aerobic stability, microbial count and in vitro ruminal fermentation, nutrients digestibility, and gas production of P. giganteum and rice straw mixed silage.

2. Materials and Methods

2.1. Silage Materials

In this study, P. giganteum was cultivated in an experimental field (86°82′ longitude and 44°19′ latitude; 6.7 °C mean temperature; 161.3 mm precipitation; 560 m altitude) with an area of 1000 m2 located in Hutubi County, Hui Autonomous Prefecture of Changji, China. The P. giganteum crop was sown on 25 May 2023, and it was harvested at the 2 m plant height (elongation stage) by cutting the whole plants (6 cm stubble height) with a reaping hook (Xinchen Machinery Co., Ltd., Jining, China). After harvesting, the P. giganteum and rice straw were chopped into small segments (1.5 to 2 cm length) by a straw cutter (Fugong Machinery Equipment Co., Ltd., Zhengzhou, China). According to the reference standard, for which the moisture content of ensiling materials is about 65%, in the current research, the mixing ratio of P. giganteum and rice straw was 73%:27%. Table 1 shows the chemical compositions of P. giganteum and rice straw.

2.2. Experimental Design

In the current experiment, the silage additives were lactic acid bacteria (Lactobacillus plantarum ≥ 1.4 × 1010 CFU/g and L. buchneri ≥ 6.0 × 109 CFU/g; Yihao Biotechnology Co., Ltd., Weifang, China) and cellulase (6000 U/g; Dongheng Huadao Biotechnology Co., Ltd., Nanning, China). Before ensiling, the raw materials (the ratio of P. giganteum to rice straw was 73%:27%) were mixed thoroughly, and then four groups were created as follows: (1) control (CON); (2) ensiled P. giganteum and rice stalk supplemented with lactic acid bacteria (LAB; the inoculated dosage was 5 mg/kg of fresh raw materials); (3) mixed silage inoculation with cellulase (CEL; the volume of the addition was 1.8 mg/kg of fresh raw materials); (4) lactic acid bacteria and cellulase treatment (LAC; the added levels were 5 mg/kg and 1.8 mg/kg of fresh raw materials, respectively).
The silage additives were mixed into distilled water (the mixed ratio of lactic acid bacteria to water was 100 mg: 500 mL; cellulase to water was 36 mg: 500 mL) and put in a sterile sprayer (Shengshi Yuanlin Technology Co., Ltd., Taizhou, China). Next, the additives were evenly sprayed on the raw materials (each group was 20 kg) layer by layer. In addition, the ensiling materials of the CON group were processed with an equivalent amount of distilled water (500 mL). Then, the P. giganteum and rice straw materials were tightly compacted and placed into a fermentation container (2L, Zhenqiang Trading Co., Ltd., Guangzhou, China) for the ensiling process in the lab. In total, there were 24 fermentation containers with 6 replicates in each group. The ensiling period lasted for 60 days. After fermentation, the silage samples were collected from each container for measurement of fermentation parameters, chemical components, microbial count, and in vitro ruminal digestibility.

2.3. Measurement of Chemical Composition and Fermentation Parameters

After ensiling, the collected silage samples were dried at 65 °C for 48 h in an air-blast drying oven (Sunnuo Instrument Technology Co., Ltd., Tianjin, China). Subsequently, the oven-dried samples were smashed using a feed mincer (Sheyan Instrument Co., Ltd., Shanghai, China) and sieved using a 1 mm screen. The contents of organic acid (OM), DM, and CP in the silage samples were determined according to the methods of AOAC [18]. The neutral detergent fiber (NDF) and acid detergent fiber (ADF) contents were determined by an Ankom fiber analyzer (A2000i, ANKOM Technology, New York, NY, USA). Furthermore, the concentration of WSC in the silage was measured by the procedures of anthrone sulfuric acid colorimetry [19].
Fresh silage samples of 20 g from each replicate were added to 180 mL of distilled water at 4 °C for 24 h. Then, the mixture was filtered through 4 layers of gauze to obtain liquid supernatant. After pH determination by a portable pH meter (Shengke Instrument Equipment Co., Ltd., Shanghai, China), total nitrogen was measured with an automatic nitrogen analyzer (HD-KN60, Horde Electronic Technology, Weifang, China), and ammonia nitrogen (NH3-N) was measured following the procedures of phenol sodium hypochlorite colorimetry [20]. Additionally, the concentrations of lactic, acetic, propionic, and butyric acids in the mixed silage samples were measured through high-performance liquid chromatography (EClassical 3200, Dalian Elite Analytical Instrument Co., Ltd., Dalian, China), referencing the procedures of Wang et al. [21].

2.4. Determination of Microbial Count and Aerobic Stability

The plate count method was utilized to enumerate the lactic acid bacteria, coliform bacteria, aerobic bacteria, mold, and yeast in the silage mixed with P. giganteum and rice stalk. In brief, fresh silage of 10 g obtained from each fermentation container was added to 90 mL of sterile water and mixed uniformly [22]. Then, the mixtures were serially diluted to count microbial composition mentioned earlier following the procedures of Sun et al. [22].
After fermentation for 60 d, the silage was subjected to an aerobic stability experiment for 5 d, referencing the procedures of a previous study [23]. The aerobic stability time was defined as the duration over which the silage mixed with P. giganteum and rice straw maintained stability prior to reaching 2 °C above room temperature (25 ± 2.8 °C) [23]. During the process, the temperature was recorded through a multi-channel temperature recorder (TP2000, Mike Sensor Technology Co., Ltd., Hangzhou, China). Moreover, the count of lactic acid bacteria, coliform bacteria, aerobic bacteria, mold, and yeast was utilized in the spoilage indexes of the mixed silage in the present study, and the analytical method was followed from a previous study [22].

2.5. In Vitro Incubation Experiment

In total, 4 Hu sheep fitted with ruminal fistulas were utilized as the donors to provide rumen fluid. The sheep were provided with the same diet twice daily (07:30 and 17:30). Table 2 shows the feed components and nutrient levels of the diet. Ruminal fluid samples were collected from the ruminal fistulas prior to morning feeding. Immediately upon collection, the ruminal fluid was filtered with 4 layers of medical cheesecloth and then placed in a prewarmed thermos at 39 °C that was filled with CO2 for the following test.
Firstly, 0.5 g of the dried silage sample was weighed and then sealed in a filter bag. The buffer solution was prepared based on the method of Menke and Steingass [24]. Subsequently, the buffer solution was mixed with the ruminal fluid in a ratio of 2:1 to prepare the mixed culture solution. The in vitro incubation process was performed using two anaerobic fermentation flasks, and the 50 mL of mixed culture solution and the 0.5 g sample in the filter bag were added in each flask with continuous CO2 exposure. After mixing, the flasks were tightly sealed and connected to the Automated Trace Gas Recording System (MC-ABSF-II). The fermentation flasks were incubated at 39 °C for 72 h, and each treatment had 4 replicates. The gas production (GP) at different time points, including 2, 4, 8, 12, 24, 48 and 72 h, was recorded.

2.6. Analysis of In Vitro Digestibility and Ruminal Fermentation

After in vitro incubation, the fermentation flasks were separated from the gas recording system. The pH of the ruminal fluid in the flasks was determined using a portable pH meter. Then, the fluid of each flask was filtered by 4 layers of gauze to obtain the incubation fluid, which was preserved in 5 mL centrifuge tubes at −80 °C for analysis of fermentation parameters. Finally, residue from each filter bag was dried at 65 °C to a constant weight. The oven-dried samples were utilized to analyze the contents of DM, OM, CP, NDF and ADF by the methods mentioned above. The in vitro nutrients (DM, OM, CP, NDF, and ADF) their digestibility (IVDMD, IVOMD, IVCPD, IVNDFD, and IVADFD) were calculated by the nutrients loss, referencing the method described by the previous studies [22,25]. In addition, the incubation fluid was thawed and centrifuged at 15,000 rpm and 4 °C for 10 min to obtain supernatant, which was used to determine the ruminal fermentation indexes of NH3-N [20], microbial protein (MCP) [26], and volatile fatty acids (VFAs)—including acetic, propionic, and butyric acids [21].

2.7. Kinetic Parameter Calculation of In Vitro Gas Production

The cumulative GP at different time points was utilized to calculate the GP parameters using an exponential curve [27] of SAS software (version 9.4) as follows: GPt = A + B (1 − e−Ct). In this equation, “GPt” represents the cumulative GP at different times (mL/g); “A” is the GP of the rapid fermentation fraction (mL/g); “B” is the GP of the slow fermentation fraction (mL/g); “C” is the rate of GP (%/h); and “t” represents the fermentation time (h).

2.8. Statistical Analysis

The SPSS software (version 22.0) was used to analyze all data through one-way ANOVA procedures, considering each fermentation container as an experimental unit. The Duncan test was employed to evaluate the differences among the four groups. Results were presented as means and standard error of the means. The statistical significance was set at p < 0.05, and 0.05 ≤ p < 0.10 indicated a tendency.

3. Results

3.1. Chemical Composition of Mixed Silage Composed of Pennisetum giganteum and Rice Straw

The effects of additives on the chemical composition of P. giganteum and rice straw mixed silage are shown in Table 3. The contents of OM and WSC were similar (p > 0.05) among the four groups. Mixed silage inoculated with LAB and LAC significantly increased (p < 0.05) the DM content. Compared with the CON group, the NDF content of silage in the LAC group decreased (p < 0.05) by 11.89%. Likewise, the ADF concentration of the LAC group was lower (p < 0.05) than that of the CON and LAB groups, whereas an opposite tendency of CP was observed between the LAC group and these two groups.

3.2. Fermentation Parameters of Mixed Silage Composed of Pennisetum giganteum and Rice Straw

Obviously, the ratio of NH3-N to total nitrogen and the pH of CON silage were higher (p < 0.05) than those of all the additive groups (Table 4). The LAC silage showed the highest lactic acid concentration, which was increased (p < 0.05) by 36.40% and 26.64%, respectively, compared with that of the CON and CEL groups. On the contrary, the propionic acid content in the LAC group was lower (p < 0.05) than that in the CON, LAB and CEL groups. Compared with CON silage, the content of acetic acid was significantly reduced (p < 0.05) in CEL and LAC silages. No butyric acid was detected in the LAB and LAC groups. In addition, the ratio of lactic acid to acetic acid of the LAC group was higher (p < 0.05) than that of the other groups.

3.3. Microbial Count of Mixed Silage Composed of Pennisetum giganteum and Rice Straw

As illustrated in Figure 1A, the number of lactic acid bacteria of the LAB and LAC treatments was significantly elevated (p < 0.05) compared with that of the CON and CEL treatments. Conversely, silage inoculated with LAB and LAC noticeably reduced (p < 0.05) the yeast count (Figure 1B). Compared with the CON group, the mold (Figure 1C), coliform bacteria (Figure 1D), and aerobic bacteria (Figure 1E) counts in the LAB and LAC groups were significantly decreased (p < 0.05). Furthermore, the count of coliform bacteria in the LAC group was lower (p < 0.05) than that of the CEL group.

3.4. Aerobic Stability of Mixed Silage Composed of Pennisetum giganteum and Rice Straw

After aerobic exposure of mixed silage for 5 d, the LAB and LAC treatments showed an observably increased (p < 0.05) lactic acid bacteria count than the CON and CEL treatments (Figure 2A). The count of yeast in the mixed silage of inoculated treatments was lower (p < 0.05) than that of the CON group (Figure 2B). No obvious difference (p > 0.05) of aerobic bacteria count was observed among all groups (Figure 2C). The mixed silage supplemented with LAB and LAC significantly reduced (p < 0.05) the quantity of mold (Figure 2D) and coliform bacteria (Figure 2E). Compared with the CON and CEL groups, the duration of aerobic stability of the mixed silage was observably lengthened (p < 0.05) in the LAB and LAC groups (Figure 2F).

3.5. In Vitro Digestibility of Mixed Silage Composed of Pennisetum giganteum and Rice Straw

As shown in Table 5, the IVOMD of silage was similar (p > 0.05) among all the groups. However, compared with the CON and LAB groups, the IVCPD of the LAC group was increased (p < 0.05) by 9.61% and 12.60%, respectively. Mixed silage with supplementation of CEL and LAC observably increased (p < 0.05) the IVNDFD. Moreover, the IVDMD of LAC silage was slightly higher (0.05 < p < 0.10) than that of the CON group. Similarly, the IVADFD of silage in the CEL group tended to be higher (0.05 < p < 0.10) compared with the CON group.

3.6. In Vitro Gas Production of Mixed Silage Composed of Pennisetum giganteum and Rice Straw

At 2 h and 12 h, the in vitro GP of LAC silage was higher (p < 0.05) than that of CON silage (Table 6). Compared with the CON and CEL groups, mixed silage with supplementation of LAB and LAC obviously increased (p < 0.05) the GP from 4 h to 8 h. With the extension of in vitro incubation time, the GP of all the silages was gradually increased. At 24 h, the GP of LAB and LAC silages was markedly increased (p < 0.05) compared with the CON group. The GP at 48 h of the LAC group tended to be higher (0.05 < p < 0.10) than that of the CON group. No obvious difference (p > 0.05) of GP was observed at 72 h among all the treatments.

3.7. Gas Production Parameters of Mixed Silage Composed of Pennisetum giganteum and Rice Straw

The GP of the slow fermentation fraction in the CEL and LAC groups was higher (p < 0.05) than that in the CON group (Table 7). Likewise, compared with CON silage, the potential GP of LAC silage was increased (p < 0.05) by 12.35%. Furthermore, LAC silage tended to have an increased (0.05 < p < 0.10) GP of the rapid fermentation fraction compared with CON and CEL silages.

3.8. In Vitro Rumen Fermentation of Mixed Silage Composed of Pennisetum giganteum and Rice Straw

Notably, the in vitro ruminal pH (Figure 3A) as well as the butyric acid (Figure 3G) and total VFA (Figure 3D) contents of mixed silage were similar (p > 0.05) among all groups. Compared with the CON and LAB groups, the concentration of NH3-N (Figure 3B) was significantly decreased (p < 0.05) in the LAC group, whereas an opposite trend of MCP content (Figure 3C) was observed between the LAC group and these two groups. Similarly, the propionic acid content (Figure 3F) of the LAC group was increased (p < 0.05) by 18.01% and 18.66%, respectively, compared with the CON and LAB groups. The ruminal acetic acid content (Figure 3E) of LAC silage was slightly higher (0.05 < p < 0.10) than that of LAB silage. Moreover, the ratio of acetic acid to propionic acid (Figure 3H) of LAC silage tended to be lower (0.05 < p < 0.10) compared with that of CON silage.

4. Discussion

4.1. Effects of Additives on Chemical Composition of Mixed Silage

Chemical components are important indexes for determining the feed value of silage. In this study, after fermentation for 60 d, supplementation with lactic acid bacteria obviously increased the DM content of silage composed of P. giganteum and rice straw. A recent study has found that mulberry silage supplementation with lactic acid bacteria can increase the DM content [28], which was in line with our study. The reason for this improvement in silage DM content may be associated with the reduced production of effluent by the action of lactic acid bacteria, which includes approximately 8% DM of silage [29]. Nevertheless, the potential mechanism of action is worth in-depth investigation. During the fermentation process, due to the action of various microorganisms, a series of biochemical reactions will occur in the silage, which can result in nutritional loss, including CP degradation to a certain degree [30]. Cellulase can promote lactic acid bacteria fermentation to produce lactic acid by providing substrate and then inhibit the degradation of CP by some spoilage microorganisms [16]. Moreover, silage supplemented with L. plantarum can accelerate the homo-fermentation of silage and inhibit the decomposition of organic substances induced by insufficient lactic acid production, which are beneficial for maintaining the nutrient contents [31]. Our study found that mixed silage with the supplementation of two additives significantly increased CP content. A similar finding was verified in research by Cheng et al. (2020) [32], who reported that the simultaneous addition of lactic acid bacteria and cellulase increased the CP content of mixed silage composed of forage soybean and corn stover. Taken together, compared with individual P. giganteum silage, the DM content increased in silage mixed with P. giganteum and rice straw [9].
The WSC is the main substrate for lactic acid bacteria to utilize. During the ensiling process, most of the WSC was utilized by the lactic acid bacteria to produce lactic acid, thus resulting in reduced WSC concentration in mixed silage. The fiber content of rice straw and P. giganteum is high, and silage inoculated with cellulase can degrade plant fiber in order to supply available WSC for lactic acid bacteria [16]. Thus, results obtained from our experiment showed that mixed silage supplemented with cellulase decreased the NDF and ADF concentrations. A recent study has verified that wet brewer’s grains and corn stover mixed silage inoculated with cellulase significantly reduced the NDF and ADF contents [33], which was in accordance with our finding. In summary of chemical composition, the combined inoculation with additives in rice straw and P. giganteum mixed silage showed the highest DM and CP contents and the lowest NDF and ADF contents, which partly attributed to the synergistic action between the two silage additives. The adequate supply of fermentation substrate was conducive to promoting lactic acid bacteria fermentation and reducing nutrients loss, suggesting that mixed silage supplemented with two additives was more effective in preserving nutritional value.

4.2. Effects of Additives on Fermentation Parameters of Mixed Silage

A pH of less than 4.2 is a critical parameter to evaluate the fermentation quality of silage [15]. In this experiment, the silage pH in the different groups was below 4.1, which partly suggested that the P. giganteum and rice straw in the mixed silage were well preserved. The silage inoculated with different additives significantly reduced the pH, among which the LAC silage showed the lowest pH value. The reduction of silage pH is attributed to the action of lactic acid bacteria, which can utilize the WSC to ferment and produce lactic acid. Compared with the other treatments, the lactic acid bacteria and fermentation substrate were both sufficient in the LAC group, which was beneficial for reducing the silage pH. A previous study of mixed silage of whole-plant corn and peanut vines reported that simultaneous supplementation with L. plantarum and cellulase reduced the pH and promoted the lactic acid production [34], which was in line with our experiment. In addition, the content of lactic acid in the LAC group was maximum and higher than that of the CON and CEL groups. In the silage, the increased lactic acid concentration can restrain the reproduction of destructive microbes and then decrease the butyric acid production [15]. In dairy cows, a previous study demonstrated that using silage with high butyric acid concentration in the diet increased the incidence rate of subclinical ketosis, which had an adverse effect on the production performance of the animals [35]. In the current experiment, the butyric acid was detected in CON and CEL silages, indicating that the silage mixed with rice straw and P. giganteum was contaminated by spoilage bacteria, which was confirmed by microbial results. The production of propionic and butyric acids is accompanied by the consumption of metabolic energy during the fermentation of silage, and the conversion of lactic acid to butyric acid can cause the loss of DM content and reduce the silage quality [36]. Results from this study displayed that mixed silage inoculation with different additives observably decreased propionic acid concentration, and the simultaneous addition showed the minimum value.
In our experiment, compared with the CON group, the content of silage acetic acid was obviously reduced in the CEL and LAC groups. The insufficient lactic acid bacteria and fermentation substrate in CON silage may shift the fermentation type from homo-fermentation to hetero-fermentation [13], resulting in increased acetic acid concentration in P. giganteum and rice straw mixed silage. Generally, the silage NH3-N content can be used to assess the degradation of protein by enzymes and microbes during the ensiling process, and the increased ratio of NH3-N to total nitrogen reflects the excessive protein decomposition of silage [37]. In our experiment, mixed silage inoculated with additives significantly decreased the ratio of NH3-N to total nitrogen, and the combined addition of two additives displayed the lowest ratio of NH3-N to total nitrogen, which suggested that the activity of proteolytic bacteria was inhibited in LAC silage. A low pH for silage can reduce the activity of proteolytic enzymes, thus alleviating protein hydrolysis and reducing NH3-N production in silage [38]. According to our results, combined inoculation with lactic acid bacteria and cellulase maintained an optimum acid environment (pH = 3.66), which was conducive to alleviating the protein degradation of the silage and decreasing the NH3-N content, as verified by the CP result mentioned previously. In addition, the reduction of NH3-N-to-total nitrogen ratio in the LAC group might be attributed to the synergistic effect of lactic acid bacteria and cellulase in causing nitrification, which converted NH3-N into nitrate nitrogen [37]. However, more experiments should be conducted in the future to study the influence of additives on nitrogen transformation in the ensiling process of P. giganteum.

4.3. Effects of Additives on Microbial Count of Mixed Silage

In the process of silage fermentation, NH3-N production and organic acid production are associated with microbial composition [16]. Our study found that compared with CON silage, the lactic acid bacteria count was noticeably elevated in LAB and LAC silages, whereas an opposite tendency of yeast, mold, coliform bacteria, and aerobic bacteria counts was observed between the CON and lactic acid bacteria treatment groups. The reduction of harmful microbial counts may be partly attributed to the adequate supply of lactic acid bacteria and carbohydrate sources after additive supplementation. Consistent with our study, previously, an experiment reported that a combined inoculation with L. plantarum and cellulase can prevent the growth of mold, yeast, and coliform bacteria in mixed silage prepared with whole-plant corn and peanut vines [34]. After supplementation with two additives in mixed silage, enough lactic acid will be produced, and then the increased lactic acid concentration can form an acid environment, thus inhibiting the reproduction and colonization of undesirable microorganisms [33]. Moreover, in the production of butyric acid in silage, harmful microorganisms, including yeast, mold, and coliform bacteria, play an essential role by secreting amino acid decarboxylases [39]. In our experiment, the relatively increased counts of these microorganisms in CON and CEL silages caused the production of butyric acid, thus decreasing the fermentation quality of P. giganteum and rice straw mixed silage.

4.4. Effects of Additives on Aerobic Stability of Mixed Silage

After being exposed to air, the activity of aerobic bacteria in the silage will increase, and then a mass of heat will be produced due to the metabolism and consumption of nutrients by bacteria, resulting in the elevation of pH and nutritional loss [15]. Our study found that the aerobic stability time of LAC silage was maximum, which suggested that combined supplementation with additives enhanced the aerobic stability of silage. As a promoter of silage aerobic spoilage, yeast is strongly linked with the increased temperature of silage [40]. The positive influence on aerobic stability by lactic acid bacteria inoculation might be partly attributed to the reduction in yeast count. In the current study, with the exception of yeast, the counts of lactic acid bacteria, mold, and coliform bacteria were similar between the CON and CEL groups. Although the fermentation substrate was adequate in CEL silage, the number of lactic acid bacteria was not enough; thus, lactic acid production was lower—a condition which cannot effectively inhibit the growth of these microorganisms [33]. After 5 d of aerobic exposure, mixed silage inoculated with lactic acid bacteria displayed significantly reduced mold and yeast counts and an increased lactic acid bacteria count. The finding indicated that supplementation with lactic acid bacteria had the ability to inhibit the growth of harmful bacteria in the mixed silage via a maintained acidic environment within a short period after air exposure. In the production of dairy cows, coliform bacteria can increase the incidence rate of diseases such as endometritis [41]. Although the number of coliform bacteria increased after aerobic exposure, our result showed that the coliform bacteria count in the LAC group was lower than that in the CON group.

4.5. Effects of Additives on In Vitro Digestibility of Mixed Silage

In addition to fermentation characteristics and nutritional components, in vitro digestibility is a critical parameter to assess the feed value of silage. In vitro nutrients digestibility is commonly utilized to evaluate the degree of feedstuff degraded by the ruminal microbiota [42]. In our study, compared with CON silage, the IVDMD and IVCPD were increased in LAC silage, indicating an improvement in nutrients digestibility of silage treated with two additives. Consistent with our finding, an experiment in Leymus chinensis silage verified that supplementation with lactic acid bacteria and cellulase significantly increased the IVDMD and IVCPD [43]. The positive effects of combined inoculation with lactic acid bacteria and cellulase on nutrients digestibility of mixed silage might be attributed to higher lactic acid production creating an acidic environment, which then restrained the activity of spoilage bacteria and decreased nutrients degradation—conditions all conducive to improving IVDMD and IVCPD. In ruminants, the content of crude fiber in feedstuff is a vital factor influencing IVNDFD and IVADFD. Our experiment showed that the P. giganteum and rice straw mixed silage inoculated with cellulase significantly increased IVNDFD. Cellulase can change the surface structure of plant fiber in silage and weaken the structural stability of the cell wall by acting on the chemical bond between cellulose and hemicellulose—bonds which are conducive to the adhesion and degradation of ruminal microorganisms, thus increasing NDF degradation [44]. Overall, combined inoculation with lactic acid bacteria and cellulase in mixed silage displayed the optimum effects for improving in vitro nutrients digestibility.

4.6. Effects of Additives on In Vitro Gas Production of Mixed Silage

In vitro GP is not only used to evaluate the nutritional value of silage but also to reflect the fermentation degree of substrate nutrients by rumen microbiota, and it is mainly dependent on the availability of soluble fractions in silage [45]. In the current study, compared with CON silage, the mixed silage in the LAC group showed a significant increase in in vitro GP from 2 h to 48 h, as well as potential GP. The possible reason was that combined inoculation with two additives in mixed silage could supply more available fermentation substrates for the ruminal microbial community, which was verified by the chemical composition results. A previous study in forage oat silage demonstrated that lactic acid bacteria inoculation significantly increased the GP within the first 48 h [46], which was in line with our finding. The lower GP in CON silage may be partly explained by the insufficient activity of ruminal microorganisms. After inoculation with silage additives, the activity of ruminal microbiota was enhanced due to the adequate supply of available nutrients. Interestingly, the GP at 72 h did not show an obvious difference among any of the treatments, which may be because the additive inoculations tended to prefer the rapid or slow fermentation fractions in P. giganteum and rice straw mixed silage.

4.7. Effects of Additives on In Vitro Rumen Fermentation of Mixed Silage

In our study, the ruminal NH3-N concentration of the LAC group was lower than that of the CON and LAB groups after in vitro fermentation, indicating that the utilization of NH3-N was improved in mixed silage after combined supplementation with two additives. The positive influence of additives on NH3-N may be related to the regulation of additives on ruminal microbiota, which was reflected by the action of lactic acid bacteria and cellulase on promoting the bacterial growth associated with nitrogen utilization [47]. NH3-N is an important material needed by ruminal microorganisms to synthesize MCP. Intriguingly, compared with the CON group, the MCP level of the LAC group was significantly increased, which was matched to the NH3-N result. In ruminants, the increased ruminal MCP is conducive to improving CP digestibility [48]; thus, the IVCPD was increased in LAC silage. Moreover, the propionic acid concentration in the incubated ruminal fluid of LAC silage was higher than that of CON and LAB silages. As a vital energy source, glucose has an important effect on various metabolism processes of animals. In ruminants, the hepatic gluconeogenesis is the main pathway to generate glucose, and propionic acid plays an essential role in the gluconeogenesis as the precursor [49]. Thus, although the current trial was an in vitro study, the propionic acid result of our experiment, to a certain extent, indicated that combined supplementation with additives in mixed silage could provide more energy for ruminants. Also, we found that the ruminal acetate-to-propionate ratio in LAC silage was slightly lower than that in CON silage, which suggested that a propionic acid fermentation type existed in the rumen treated by LAC silage. In the rumen, propionic acid fermentation can supply more energy to promote growth and production performance of ruminants [48]. The microbiota in the rumen has vital function in NH3-N utilization, MCP synthesis, and VFA production. A recent study found that cellulase and Lactiplantibacillus plantarum supplementation can improve in vitro rumen fermentation and nutrients degradation of Caragana korshinskii silage by regulating the microbial composition [50]. In future studies, the effects of P. giganteum and rice straw mixed silage supplementation with lactic acid bacteria and cellulase on ruminal microbiota of different growth stages of ruminants should be investigated in depth by feeding experiments.

5. Conclusions

The findings obtained from this experiment provide evidence that P. giganteum and rice straw mixed silage supplementation with lactic acid bacteria and cellulase improves chemical composition and fermentation quality to some extent. Combining the two additives shows maximum improvement in silage quality, which is reflected by the increase in CP, DM, and lactic acid contents, reduction of NH3-N/total nitrogen as well as yeast, mold and coliform bacteria counts, and improvement of aerobic stability. Additionally, the in vitro nutrients digestibility, gas production, and ruminal fermentation are improved by mixed silage composed of P. giganteum and rice straw and by supplementation with lactic acid bacteria and cellulase. According to the findings mentioned previously, combined inoculation with lactic acid bacteria and cellulase is an effective strategy to improve the quality of P. giganteum and rice straw mixed silage.

Author Contributions

Conceptualization, J.M. and S.G.; methodology, J.M., L.L., Y.L. and R.G.; software, B.W. and C.W.; validation, L.L., Y.F. and C.D.; formal analysis, C.D. and C.W.; investigation, J.M., L.L., Y.L., B.W., Y.F. and R.G.; resources, R.G. and S.G.; data curation, J.M. and S.G.; writing—original draft preparation, J.M.; writing—review and editing, J.M., R.G. and S.G.; visualization, C.D. and C.W.; supervision, S.G.; project administration, J.M. and S.G.; funding acquisition, J.M. and S.G. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Innovative Training Program for College Students (grant number S202410566028) and the Program for Scientific Research Start-up funds of Guangdong Ocean University (grant number 060302052308).

Institutional Review Board Statement

The animal study protocol was approved by the Institutional Animal Care and Use Committee of Guangdong Ocean University (Zhanjiang, Guangdong, China; Approval Code: SYXK-2023-108).

Data Availability Statement

The data included in this research are available upon request from the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Effects of additives on microbial count of mixed silage. (A) Lactic acid bacteria; (B) yeast; (C) mold; (D) coliform bacteria; (E) aerobic bacteria. FM, fresh matter; CFU, colony forming unit. CON, control group with no silage additive; LAB, P. giganteum and rice straw mixed silage with inoculation of lactic acid bacteria 5 mg/kg; CEL, silage with inoculation of cellulase 1.8 mg/kg; LAC, silage with inoculation of lactic acid bacteria 5 mg/kg and cellulase 1.8 mg/kg. Columns with different lowercase letters differ significantly (p < 0.05).
Figure 1. Effects of additives on microbial count of mixed silage. (A) Lactic acid bacteria; (B) yeast; (C) mold; (D) coliform bacteria; (E) aerobic bacteria. FM, fresh matter; CFU, colony forming unit. CON, control group with no silage additive; LAB, P. giganteum and rice straw mixed silage with inoculation of lactic acid bacteria 5 mg/kg; CEL, silage with inoculation of cellulase 1.8 mg/kg; LAC, silage with inoculation of lactic acid bacteria 5 mg/kg and cellulase 1.8 mg/kg. Columns with different lowercase letters differ significantly (p < 0.05).
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Figure 2. Effects of additives on aerobic stability of mixed silage. (A) Lactic acid bacteria; (B) yeast; (C) aerobic bacteria; (D) mold; (E) aerobic bacteria; (F) aerobic stability time. FM, fresh matter; CFU, colony forming unit. CON, control group with no silage additive; LAB, P. giganteum and rice straw mixed silage with inoculation of lactic acid bacteria 5 mg/kg; CEL, silage with inoculation of cellulase 1.8 mg/kg; LAC, silage with inoculation of lactic acid bacteria 5 mg/kg and cellulase 1.8 mg/kg. Columns with different lowercase letters differ significantly (p < 0.05).
Figure 2. Effects of additives on aerobic stability of mixed silage. (A) Lactic acid bacteria; (B) yeast; (C) aerobic bacteria; (D) mold; (E) aerobic bacteria; (F) aerobic stability time. FM, fresh matter; CFU, colony forming unit. CON, control group with no silage additive; LAB, P. giganteum and rice straw mixed silage with inoculation of lactic acid bacteria 5 mg/kg; CEL, silage with inoculation of cellulase 1.8 mg/kg; LAC, silage with inoculation of lactic acid bacteria 5 mg/kg and cellulase 1.8 mg/kg. Columns with different lowercase letters differ significantly (p < 0.05).
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Figure 3. Effects of additives on in vitro rumen fermentation parameters of mixed silage. (A) pH; (B) NH3-N; (C) MCP; (D) Total VFA; (E) acetic acid; (F) propionic acid; (G) butyric acid; (H) acetic acid-to-propionic acid ratio. NH3-N, ammonia nitrogen; MCP, microbial protein; VFA, volatile fatty acid. CON, control group with no silage additive; LAB, P. giganteum and rice straw mixed silage with inoculation of lactic acid bacteria 5 mg/kg; CEL, silage with inoculation of cellulase 1.8 mg/kg; LAC, silage with inoculation of lactic acid bacteria 5 mg/kg and cellulase 1.8 mg/kg. Columns with different lowercase letters differ significantly (p < 0.05).
Figure 3. Effects of additives on in vitro rumen fermentation parameters of mixed silage. (A) pH; (B) NH3-N; (C) MCP; (D) Total VFA; (E) acetic acid; (F) propionic acid; (G) butyric acid; (H) acetic acid-to-propionic acid ratio. NH3-N, ammonia nitrogen; MCP, microbial protein; VFA, volatile fatty acid. CON, control group with no silage additive; LAB, P. giganteum and rice straw mixed silage with inoculation of lactic acid bacteria 5 mg/kg; CEL, silage with inoculation of cellulase 1.8 mg/kg; LAC, silage with inoculation of lactic acid bacteria 5 mg/kg and cellulase 1.8 mg/kg. Columns with different lowercase letters differ significantly (p < 0.05).
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Table 1. Chemical composition of Pennisetum giganteum and rice straw (%, DM basis).
Table 1. Chemical composition of Pennisetum giganteum and rice straw (%, DM basis).
ItemsOMDMNDFADFCPWSC
Pennisetum giganteum88.1715.1356.9736.8513.286.76
Rice straw87.7589.6771.0546.725.172.42
OM, organic matter; DM, dry matter; NDF, neutral detergent fiber; ADF, acid detergent fiber; CP, crude protein; WSC, water soluble carbohydrate.
Table 2. Feed ingredients and nutrient levels of the basal diet (DM basis).
Table 2. Feed ingredients and nutrient levels of the basal diet (DM basis).
Ingredients Nutrient Levels
Corn32.97CP (%)13.60
Cottonseed meal3.88NDF (%)37.42
Wheat bran6.08ADF (%)22.06
Corn gluten meal5.16Ca (%)0.76
Soybean meal3.12P (%)0.41
NaCl0.50ME 2 (MJ/kg)8.67
Limestone1.09
Premix 12.20
Alfalfa hay10.05
Corn straw34.95
DM, dry matter; CP, crude protein; NDF, neutral detergent fiber; ADF, acid detergent fiber; ME, metabolizable energy. 1 The premix provided the following per kilogram of diet: Fe 270 mg, Zn 190 mg, Mn 180 mg, Cu 70 mg, I 16 mg, Se 11 mg, Co 17 mg, VA 350,000 IU, VD 110,000 IU, and VE 3000 IU. 2 ME was a calculated value, and other nutrient levels were measured values.
Table 3. Effects of additives on chemical composition of mixed silage (%, DM basis).
Table 3. Effects of additives on chemical composition of mixed silage (%, DM basis).
ItemsGroupsSEMp-Value
CONLABCELLAC
DM22.76 b26.31 a23.27 b27.49 a0.7190.039
NDF59.38 a57.72 ab53.62 ab52.32 b1.0590.046
ADF39.31 a38.04 ab35.27 bc34.10 c0.7320.031
OM86.6585.2489.0488.270.6260.133
CP7.02 b7.13 b7.55 ab8.03 a0.1240.006
WSC2.772.282.672.360.1170.396
DM, dry matter; NDF, neutral detergent fiber; ADF, acid detergent fiber; OM, organic matter; CP, crude protein; WSC, water soluble carbohydrate; SEM, standard error of the mean. CON, control group with no silage additive; LAB, P. giganteum and rice straw mixed silage with inoculation of lactic acid bacteria 5 mg/kg; CEL, silage with inoculation of cellulase 1.8 mg/kg; LAC, silage with inoculation of lactic acid bacteria 5 mg/kg and cellulase 1.8 mg/kg. Means with different superscript letters in the same row differ significantly (p < 0.05).
Table 4. Effects of additives on fermentation parameters of mixed silage.
Table 4. Effects of additives on fermentation parameters of mixed silage.
ItemsGroupsSEMp-Value
CONLABCELLAC
pH4.10 a3.76 b3.80 b3.66 b0.0550.020
Lactic acid (%, DM)4.67 c5.74 ab5.03 bc6.37 a0.1980.004
Acetic acid (%, DM)1.45 a1.22 ab1.17 b1.02 b0.0500.013
Propionic acid (%, DM)0.042 a0.022 b0.019 b0.009 c0.003<0.001
Butyric acid (%, DM)0.004ND0.001ND0.0010.075
Lactic acid/Acetic acid3.25 c4.78 b4.38 bc6.48 a0.3280.001
NH3-N/total nitrogen5.47 a4.25 b4.16 b3.76 b0.2190.022
DM, dry matter; ND, not detected; NH3-N, ammonia nitrogen; SEM, standard error of the mean. CON, control group with no silage additive; LAB, P. giganteum and rice straw mixed silage with inoculation of lactic acid bacteria 5 mg/kg; CEL, silage with inoculation of cellulase 1.8 mg/kg; LAC, silage with inoculation of lactic acid bacteria 5 mg/kg and cellulase 1.8 mg/kg. Means with different superscript letters in the same row differ significantly (p < 0.05).
Table 5. Effects of additives on in vitro digestibility of mixed silage (%).
Table 5. Effects of additives on in vitro digestibility of mixed silage (%).
ItemsGroupsSEMp-Value
CONLABCELLAC
IVDMD48.8253.1250.1454.020.8400.084
IVOMD54.7152.2951.4854.091.4720.870
IVCPD54.95 b53.49 b57.31 ab60.23 a0.9410.048
IVNDFD34.31 b32.99 b39.38 a38.64 a0.7910.002
IVADFD28.1629.2232.5231.670.7110.092
IVDMD, in vitro dry matter digestibility; IVOMD, in vitro organic matter digestibility; IVCPD, in vitro crude protein digestibility; IVNDFD, in vitro neutral detergent fiber digestibility; IVADFD, in vitro acid detergent fiber digestibility; SEM, standard error of the mean. CON, control group with no silage additive; LAB, P. giganteum and rice straw mixed silage with inoculation of lactic acid bacteria 5 mg/kg; CEL, silage with inoculation of cellulase 1.8 mg/kg; LAC, silage with inoculation of lactic acid bacteria 5 mg/kg and cellulase 1.8 mg/kg. Means with different superscript letters in the same row differ significantly (p < 0.05).
Table 6. Effects of additives on in vitro gas production of mixed silage (mL/g).
Table 6. Effects of additives on in vitro gas production of mixed silage (mL/g).
ItemsGroupsSEMp-Value
CONLABCELLAC
2 h14.22 b16.25 ab14.90 ab16.88 a0.3880.047
4 h18.18 b21.96 a17.69 b21.87 a0.7020.031
8 h26.24 b31.71 a27.26 b32.04 a0.8430.013
12 h33.32 b38.96 ab35.03 ab40.63 a1.0620.041
24 h48.38 b56.54 a51.21 ab55.78 a1.1410.022
48 h68.0974.3772.6476.171.3010.085
72 h73.9776.8775.3180.691.4450.407
SEM, standard error of the mean. CON, control group with no silage additive; LAB, P. giganteum and rice straw mixed silage with inoculation of lactic acid bacteria 5 mg/kg; CEL, silage with inoculation of cellulase 1.8 mg/kg; LAC, silage with inoculation of lactic acid bacteria 5 mg/kg and cellulase 1.8 mg/kg. Means with different superscript letters in the same row differ significantly (p < 0.05).
Table 7. Effects of additives on gas production parameters of mixed silage.
Table 7. Effects of additives on gas production parameters of mixed silage.
ItemsGroupsSEMp-Value
CONLABCELLAC
A (mL/g)1.261.501.251.670.0690.088
B (mL/g)68.28 b72.68 ab73.93 a76.47 a1.0470.032
A + B (mL/g)69.54 b74.18 ab75.18 ab78.13 a1.0800.030
C (%/h)0.0750.0670.0620.0600.0040.596
A, GP of rapid fermentation fraction; B, GP of slow fermentation fraction; A + B, potential GP; C, rate of GP; GP, gas production; SEM, standard error of the mean. CON, control group with no silage additive; LAB, P. giganteum and rice straw mixed silage with inoculation of lactic acid bacteria 5 mg/kg; CEL, silage with inoculation of cellulase 1.8 mg/kg; LAC, silage with inoculation of lactic acid bacteria 5 mg/kg and cellulase 1.8 mg/kg. Means with different superscript letters in the same row differ significantly (p < 0.05).
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MDPI and ACS Style

Ma, J.; Lin, L.; Lu, Y.; Weng, B.; Feng, Y.; Du, C.; Wei, C.; Gao, R.; Gan, S. The Influence of Silage Additives Supplementation on Chemical Composition, Aerobic Stability, and In Vitro Digestibility in Silage Mixed with Pennisetum giganteum and Rice Straw. Agriculture 2024, 14, 1953. https://doi.org/10.3390/agriculture14111953

AMA Style

Ma J, Lin L, Lu Y, Weng B, Feng Y, Du C, Wei C, Gao R, Gan S. The Influence of Silage Additives Supplementation on Chemical Composition, Aerobic Stability, and In Vitro Digestibility in Silage Mixed with Pennisetum giganteum and Rice Straw. Agriculture. 2024; 14(11):1953. https://doi.org/10.3390/agriculture14111953

Chicago/Turabian Style

Ma, Jian, Lu Lin, Yuezhang Lu, Beiyu Weng, Yaochang Feng, Chunmei Du, Chen Wei, Rui Gao, and Shangquan Gan. 2024. "The Influence of Silage Additives Supplementation on Chemical Composition, Aerobic Stability, and In Vitro Digestibility in Silage Mixed with Pennisetum giganteum and Rice Straw" Agriculture 14, no. 11: 1953. https://doi.org/10.3390/agriculture14111953

APA Style

Ma, J., Lin, L., Lu, Y., Weng, B., Feng, Y., Du, C., Wei, C., Gao, R., & Gan, S. (2024). The Influence of Silage Additives Supplementation on Chemical Composition, Aerobic Stability, and In Vitro Digestibility in Silage Mixed with Pennisetum giganteum and Rice Straw. Agriculture, 14(11), 1953. https://doi.org/10.3390/agriculture14111953

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