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Article

Effects of Rumen-Degradable Starch Levels on In Vitro Rumen Fermentation and Microbial Protein Synthesis in Alfalfa Silage

by
Wenliang Guo
1,
Yulan Liu
2,
Meila Na
1,
Yu Zhang
1 and
Renhua Na
1,*
1
College of Animal Science, Inner Mongolia Agricultural University, Hohhot 010018, China
2
Inner Mongolia Zhamuqin Agriculture and Animal Husbandry Technology Co., Ltd., Ulanhot 029400, China
*
Author to whom correspondence should be addressed.
Fermentation 2025, 11(2), 106; https://doi.org/10.3390/fermentation11020106
Submission received: 4 February 2025 / Revised: 16 February 2025 / Accepted: 18 February 2025 / Published: 19 February 2025
(This article belongs to the Section Microbial Metabolism, Physiology & Genetics)
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Abstract

:
Alfalfa silage has a high proportion of rumen-degradable protein content. Increasing dietary rumen-degradable starch (RDS) can enhance ruminal microbial protein synthesis. This study was conducted to investigate the influence of RDS levels in substrates with alfalfa silage on in vitro rumen fermentation and nitrogen (N) utilization. Rumen fluid was collected and dispensed into anaerobic fermentation bottles, each containing 1 g of substrate and 60 mL of rumen fluid–buffer mixture. The substrate was composed of 40% alfalfa silage and five different RDS levels: 14.85% RDS, 16.40% RDS, 18.67% RDS, 20.21% RDS, and 21.62% RDS. For each RDS level, three replicates were prepared. Each substrate was then incubated at 39 °C for 3, 6, 12, and 24 h. After incubation, the following parameters were measured: gas production, pH, α-amylase activity, ammonia nitrogen (NH3-N), bacterial protein (BCP), and short-chain fatty acid (SCFA) concentrations were measured. Total gas production increased linearly with increasing RDS levels from 3 to 10 h of incubation (p < 0.01), with no difference observed among five levels after 11 h. At 3 h of incubation, pH decreased linearly with increasing RDS levels (p < 0.05). BCP concentrations and α-amylase activity increased linearly or quadratically with increasing RDS levels (p < 0.01), while the R4 group had the highest concentrations of BCP and the R5 group had the highest activity of α-amylase (p < 0.01). At 6 h of incubation, the NH3-N concentration decreased linearly or quadratically with increasing RDS levels (p < 0.05), and the α-amylase activity, acetate, propionate, and total SCFA concentrations increased linearly (p < 0.01). The R4 group had the highest activity of α-amylase (p < 0.01), and the R5 group had the highest concentrations of acetate (p < 0.05) and propionate (p < 0.01). At 12 h of incubation, BCP, NH3-N, and propionate concentrations, as well as α-amylase activity, increased linearly or quadratically with increasing RDS levels (p < 0.05). At 24 h of incubation, the α-amylase activity increased linearly with increasing RDS levels (p < 0.05). The highest multiple-factor associative effects index was observed in the 20.21% RDS substrate, indicating that an RDS level of 20.21% in the alfalfa silage substrate resulted in a desirable rumen N utilization.

1. Introduction

Alfalfa is an important roughage for ruminant feed due to its excellent amino acid profile and low lignocellulose content. Ensiling is one of the most frequent forms of processing alfalfa [1,2,3]. However, alfalfa typically has higher crude protein content than other forages. The proportion of protein in alfalfa silage in the form of non-protein nitrogen (NPN) is higher due to proteolysis during preservation [4]. The ammonia-N pool and non-ammonia NPN pool are substantially larger in alfalfa silage than in alfalfa hay, the proportion of rumen-degradable protein (RDP) is increased, and the true protein value is lost [5]. Feeding more alfalfa silage results in inefficient N utilization and increased urinary-N excretion [6]. A direct comparison of alfalfa hay and silage as sole forage sources for lactating cows revealed that N utilization efficiency was lower in silage than in hay [5]. When cows were fed a greater proportion of alfalfa silage, milk yield, total microbial protein, total essential amino acid flow at the omasal canal, and total nitrogen excretion increased [7].
Rumen microorganisms can synthesize microbial protein with high efficiency using NPN. Amino acid- and peptide-N are preferred N sources for ruminal microorganisms, and sufficient energy is critical for ruminal bacterial protein (BCP) synthesis. The inclusion of ground high-moisture corn (HMC) in dairy cow diets stimulated NH3 uptake in vitro and stimulated milk protein secretion relative to the inclusion of unground HMC [8]. The inclusion of 40% ground HMC in dairy cow diets increased BCP synthesis in the rumen relative to 24% ground HMC [9]. Finely ground corn in dairy cow diets increased ruminal starch digestion and tended to enhance microbial nitrogen flow in the duodenum compared to medium-grind corn [10]. These results suggest that enhancing the availability of ruminal fermentable energy might be an effective strategy to increase microbial capture of RDP from alfalfa silage, reducing N excretion.
Starch is a major component of cereals and the primary energy source for the fattening of ruminants. Corn and wheat have been important sources of diet for ruminant and non-ruminant animals due to their high production and starch content [11,12]. Wheat, a cereal with a high rumen-degradable starch (RDS) content, is commonly included in Australian dairy cow diets [13,14,15]. The starch degradation rate of wheat was 24.8% higher than that of corn [16]. Increasing dietary RDS has been shown to optimize digestion of carbohydrates and protein [13,17,18], reduce methane emissions [19], and increase protein flow to the small intestine [15,20]. Several studies with in vitro rumen fermentation have reported the optimal RDS levels in diets [21,22]. However, research on the alfalfa silage diet is still lacking. We hypothesized that increasing starch rumen degradability of finishing diets would improve the nitrogen utilization rate of alfalfa silage and stimulate synthesis of microbial protein in the rumen. Therefore, the main aim of the present research was to investigate the dynamic changes of different RDS levels on in vitro fermentation of alfalfa silage.

2. Materials and Methods

2.1. Composition and Nutritional Level of Experimental Diet

The alfalfa was collected in the Tumochuan Plain (111°30′ E, 40°45′ N), located in Hohhot, Inner Mongolia, China. The Tumochuan Plain has a temperate continental climate with an annual average temperature ranging from 2 to 5 °C. The hottest month is July, with an average temperature of 20.5 °C. Precipitation is mainly concentrated in July and August, with an annual average precipitation of 379.37 mm and an annual average evaporation of 1851 mm.
The alfalfa (cultivar WL354) was grown in a 2000 m2 area, with rows spaced 30–40 cm apart. Phosphorus and potassium fertilizers were applied before sowing alfalfa, and no additional fertilizer was applied during its growing period. Irrigation was conducted after each harvest. The second cut (14 days after first cut) of pure alfalfa was harvested using a mower at the early flowering stage, at a height of 6 cm from the ground. The alfalfa was chopped into 2.0 ± 0.1 cm size and then equally spread on a white plastic sheet in the shade for a low-intensity wilting treatment until dry matter (DM) concentrations of 400 g/kg were reached, then packed into the white plastic sheet, vacuum-sealed, and stored. Corn, wheat, corn stalk, and other ingredients were obtained from a local feed mill. All ingredients were then dried in a forced air oven at 65 °C for 48 h and then weighed to determine DM content. They were then ground in a mill to pass through a 1 mm sieve for later analysis. Total nitrogen (TN) content was determined using the Kjeldahl nitrogen apparatus (Hanon, K9840, Jinan, Shandong, China). Crude protein (CP) was determined by multiplying the TN by 6.25. Neutral detergent fiber (NDF) and acid detergent fiber (ADF) were measured using an ANKOM fiber analyzer (Hanon, F800, Jinan, Shandong, China) [3]. Ether extract (EE) was determined using hexane in a fat extractor (Hanon, SOX406, Jinan, Shandong, China). Digestible energy (DE) was calculated by referring to the raw materials [23]. The starch was determined with commercial kits (Nanjing Jiancheng Institute of Bioengineering, Nanjing, China). The nutrient composition of the substrate raw materials is shown in Table 1. Five RDS levels of substrates used in this study were prepared according to the proportion of wheat starch to corn starch in the diet. The chemical composition and nutritional levels of each substrate are listed in Table 2.

2.2. In Vitro Experiment

Rumen fluid was obtained from six Hu sheep (2.5 years old, body weight of 42 ± 3 kg) housed at the experimental farm located in Hohhot. Each sheep was fed ad libitum a total mixed ration containing(per kg DM) corn stalk (100 g), alfalfa silage (400 g), corn (300 g), and concentrate (200 g), with the following nutrient levels: CP: 15.03%, NDF: 29.12%, and EE: 4.50%. Rumen fluid was collected before the morning feeding using a tube attached to a vacuum pump and strained through four layers of gauze into a pre-warmed flask at 39 °C filled with carbon dioxide, sealed at the top, and taken to the laboratory.
Five levels of substrates with different RDS contents (14.85% RDS, R1; 16.40% RDS, R2; 18.67% RDS, R3; 20.21% RDS, R4; and 21.62% RDS, R5) were incubated in triplicate for 3, 6, 12, and 24 h, respectively. Real-time monitoring of gas production was conducted, resulting in a total of 75 incubation bottles being used (i.e., 5 RDS × 4 sampling times × 3 replicates + 5 RDS × 1 gas production device × 3 replicates). The buffer solution was prepared according to the method of Menke et al. [24]. The rumen fluid was mixed with a reduced, pre-warmed buffer at a ratio of 1:2 under continuous flushing with CO2. After equilibrating the solution with CO2, a 60 mL aliquot of rumen fluid–buffer mixture was added to 100 mL incubation bottles containing 1.0 g of the substrate. The bottles were closed with butyl rubber stoppers and randomly placed in a constant-temperature oscillation incubator (120 r/min) at 39 °C (Herrytech, HT-1102XS-J, Shanghai, China). Of the 75 bottles, 15 bottles (5 RDS × 3 replicates) were connected to the gas measurement system (AGRS, Beijing, China) via a needle, and gas production was recorded automatically every minute. All incubation bottles were carried out simultaneously. All procedures described above were conducted under anaerobic conditions. Three bottles of each RDS level were removed from the incubator at 3, 6, 12, and 24 h of incubation, and were placed in ice water to stop fermentation. Subsequently, the pH was measured using a portable pH meter (Lichen, PH-10, Shenzhen, China) [25]. The mixed fluid was then transferred into microcentrifuge tubes and centrifuged (3000 r/min−1) to separate the supernatant (Kaidasy, KL04A, Changsha, Hunan, China), and stored under −20 °C for use in measuring short-chain fatty acids (SCFA), including acetate, propionate, butyrate, isbutyric, valerate, and isovalerate. The SCFA content was analyzed by gas chromatography (6890N; Agilent, Santa Clara, CA, USA) [26]. The ammonia nitrogen (NH3-N) and bacterial protein (BCP) contents were assessed by spectrometer using colorimetry [27] and Coomath bright blue process [28], respectively. Gas production was calculated based on Brewster’s method [29]. α-amylase activity was determined using commercial kits (Nanjing Jiancheng Institute of Bioengineering, Nanjing, China). Dietary RDS was calculated using the following Formula (1) [30]. The single-factor associative effects index (SFAEI) and multiple-factor associative effects index (MFAEI) were calculated according to Equations (2) and (3) [31].
RDS = ∑ i = 1 n p i × ERD i
where Pi represents the proportion of dietary starch of feed i in the diet, ERDi represents starch-effective degradability of feed i, and n is the number of feeds in the diet. The ERD parameters were referred to Noblet et al. [23].
SFAEI = Σnm = 1(A2m − A1m)/A2m/n
m is each incubation time (i.e., m = 1, 2, 3, and 4); n is the number of incubation times (n = 4); A1m is the value of each single index in the control group (R1) at different sampling times. A2m is the value of each single index in the experimental group (R2, R3, R4, and R5) at different culture times.
MFAEI = the sum of each single − factor associative effect value
In our study, MFAEI is the sum of the four SFAEI values of TSCFA, α-amylase, GP, and MCP.

2.3. Data Processing and Analysis

The data were first processed by Microsoft Excel 2016 and then analyzed by one-way ANOVA using statistical analysis software SAS (Version 9.2, SAS Institute Inc., Cary, NC, USA). Each individual bottle was an experimental unit for all collected data. Data were presented as averages. Duncan’s multiple range test (DMRT) was conducted to evaluate the differences among treatments. Meanwhile, regression (REG) analysis was conducted to evaluate the linear and quadratic effects of the increasing levels of RDS on the fermentation parameters, considered significant at p < 0.05 and extremely significant at p < 0.01.

3. Results

3.1. Effects of RDS Levels on Total Gas Production During In Vitro Rumen Fermentation of Alfalfa Silage

The total gas production during in vitro rumen fermentation of the different substrates is shown in Figure 1. The total gas production had no significant difference among groups at 2 h, but it increased linearly with increasing RDS levels from 3 to 10 h of incubation (p < 0.01). However, no significant difference was observed among the five groups from 11 to 25 h of incubation.

3.2. Effects of RDS Levels In Vitro Rumen Fermentation Parameters of Alfalfa Silage

The effects of RDS levels on in vitro rumen fermentation parameters of alfalfa silage are shown in Table 3 and Table 4. At 3 h of incubation, pH decreased linearly with increasing RDS levels (p < 0.05). BCP concentrations and α-amylase activity increased linearly or quadratically with increasing RDS levels (p < 0.01), while the R4 group had the highest concentrations of BCP and the R5 group had the highest activity of α-amylase (p < 0.01). At 6 h, NH3-N concentrations decreased linearly or quadratically with increasing RDS levels (p < 0.01), and α-amylase activity, acetate, propionate, and TSCFA concentrations increased linearly or quadratically with increasing RDS levels (p < 0.01). Specifically, the R4 group had the highest activity of α-amylase (p < 0.01), and the R5 group had the highest concentrations of acetate (p < 0.05) and propionate (p < 0.01). At 12 h, BCP, NH3-N, and propionate concentrations increased linearly with increasing RDS levels (p < 0.05), while α-amylase activity increased quadratically (p < 0.05). At 24 h, α-amylase activity showed a linear increase with RDS levels (p < 0.05). No significant difference was observed in the content of other variables.

3.3. The Effects of RDS Levels on In Vitro Rumen Fermentation SFAEI and MFAEI of Alfalfa Silage

The effects of RDS levels on in vitro rumen fermentation SFAEI and MFAEI of alfalfa silage are shown in Figure 2. The SFAEI of TSCFA, α-amylase, GP, and BCP increased linearly with increasing RDS levels, and the highest BCP, GP, α-amylase, and TSCFA were in R4, R3, R3, and R5 substrate, respectively. The highest MFAEI was in the R4 substrate.

4. Discussion

Alfalfa silage technology reduces leaf loss and increases palatability. Enhancing nitrogen retention may alter the nitrogen composition of silage. Specifically, the proportions of NPN to total N in the silage are 51.9%, with NH3-N, free amino acid N, and unidentified NPN accounting for 7.8%, 29.2%, and 14.9%, respectively. The rumen digests rapidly [32]. Synchronizing the starch degradation rate in the rumen can maximize the growth of rumen microorganisms, thereby enhancing the nitrogen efficiency of alfalfa silage [33]. However, the results were not consistent across different substrates [34]. Here, we evaluated the fermentation parameters of alfalfa silage with different RDS levels through in vitro experiments, and found that the rumen N utilization was high, with the optimal level of dietary RDS found to be 20.21%.
Gas production data can predict in vivo rumen OM digestibility. In our study, with increasing RDS levels, total gas production increased linearly or quadratically at 3 to 9 h of incubation, consistent with previous in vitro studies [35]. Xu et al. [21] observed that as the ratio of wheat in the substrate increased, the rate and extent of gas production also increased linearly in vitro. Accordingly, Calabro et al. [36], comparing fresh and dried silage found that higher availability of energy and N improves the microbial growth, mainly at the beginning of incubation, resulting in more rapid fermentation and increased gas production. This suggests that increasing RDS levels enhance the fermentation rate in the rumen. The starch granules in corn endosperm are encompassed by phosphorus-associated components more tightly than the wheat endosperm [21], and wheat has a lower amylose-to-amylopectin ratio than corn [37]. Therefore, the proportion of RDS has positive associative effects on the GP rate. However, after 9 h of incubation, no difference in GP was observed among the groups, which can be attributed to the similar starch concentration in each group.
Substrates’ pH decreased linearly with increasing RDS levels, suggesting that more rapid degradation of RDS produces lactic acid, formate, and acetic acid. Therefore, high RDS levels are also commonly used as a raw material to trigger subacute ruminal acidosis [38]. Similarly, Russo [39] reported a decrease in mean, minimum, and maximum pH in the rumen with an increase of crushed wheat levels in lactating cow diets. In our experiment, the α-amylase activity and BCP concentration increased linearly in substrates with increasing RDS levels. Similarly, Shen [26] reported an increase in amyloglucosidase in the rumen with increasing RDS levels in dairy goat diets. Enzymatic starch availability is thought to reflect the rate of ruminal starch fermentation [40]. Starch digestion in the rumen is affected by enzyme activity, which is affected by rumen microbial activity [38]. In the present study, BCP concentration after 3 h of incubation increased linearly with increasing RDS levels, while NH3-N concentrations after 6 h of incubation decreased linearly. This result indicated that rumen microbial growth is energy-dependent, and the sequence of N utilization is polypeptide, amino acid, and ammonia nitrogen successively. The different RDS levels had only a minor impact on NH3-N and BCP after 24 h of incubation, which may be due to substrate starch energy depletion and pH being too low, causing microbial activity to stop. Similarly, the increased starch degradability of steam-flaked corn (SFC) decreased ruminal NH3-N and blood urea concentration in cows receiving SFC compared to cows fed dry ground corn [41]. The substrate with a concentrate-to-forage ratio of 5:5 and a wheat-to-corn ratio of 2:8 had the highest BCP and lowest NH3-N concentration, indicating that different proportions of structural carbohydrates and nitrogen sources in the diet would also affect the optimal grain ratio [21]. In this study, it was observed that nitrogen utilization by rumen bacteria increased to varying degrees with increasing RDS levels, with 20.21% RDS exhibiting the best effects.
In the rumen, starch is degraded mainly by the α-amylases from rumen microorganisms to release maltose, glucose, and other disaccharides, which are then converted into SCFA and methane by glycolysis. Acetate and propionate are the main end products of rumen microbial fermentation, and forming propionate consumes H+ to produce more ATP compared to acetate, resulting in less methane loss and higher energy efficiency. In the present study, the substrate’s acetate, propionate, and TSCFA increased linearly with increasing RDS levels after 6 h of incubation, which supports the idea that the high RDS proportion tends to favor propionic fermentation and has a greater in vitro antimethanogenic effect [42]. A series of studies showed that a reduction of methane emissions with increased rumen-degradable starch was also observed in cows, with increased rumen propionate content [25,41,43] and TSCFA content [44]. The increased propionate in the substrate with a relatively high RDS proportion may be attributed to the quick accumulation of succinate, which is a precursor of propionates [33], and the propionate-producing bacteria are more active under low pH [45]. In addition, another experiment also showed a strong negative correlation (r = −0.95) between total ruminal SCFA concentration and pH [33]. However, despite limited differences in the acetate/propionate ratio between RDS and alfalfa silage in the present study, TSCFA in the incubation medium increased with increasing RDS levels in the substrate, indicating that alfalfa silage provided a good nitrogen source for microorganisms, produced more amylase, and therefore increased the yield of TSCFA. In addition, even though the release of CO2 from microbial metabolism is associated with acetate production, the slight difference in the acetate/propionate ratio supports the significant increase of gas production with the level of RDS in the substrate [36]. However, after 12 h, the SCFA production from the five substrates was not significantly different, which is similar to previous research [46]. In addition, we evaluated the MFAEI of substrates with different RDS levels. It was found that the MFAEI of the substrate with 20.21% RDS was optimal. It is worth noting that high production of fermentation acid will impair rumen digestive function and animal health. Moreover, diets with high RDS levels for lactating goats resulted in little difference in DMI and milk production but caused potential risks of subacute ruminal acidosis [47]. However, the inflammatory response was not evaluated in the current study.

5. Conclusions

In the current study, the total gas production, BCP, α-amylase activity, and TSCFA increased with increasing RDS levels during the period from 3 to 12 h of incubation; NH3-N and pH were reduced linearly; and the highest BCP content and MFAEI were observed in the 20.21% RDS substrate. Therefore, an RDS level of 20.21% in the alfalfa silage diet resulted in a desirable rumen N utilization.

Author Contributions

R.N. and Y.Z. designed this experiment. W.G. and M.N. performed the animal feeding experiments, measured and acquired the data, and participated in results interpretation and statistical analysis. W.G. wrote and edited the manuscript. Y.L. provided financial support. R.N. reviewed and revised this paper. R.N. supervised all processes from performing the experiment to writing the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Project on “Standardization of Healthy Livestock and Poultry Breeding and Harmless Treatment of Livestock and Poultry Waste in Horqin Youyi Zhongqi (2022YFXZ0027)”, the “Innovative Research Team in Universities of Inner Mongolia Autonomous Region (NMGIRT2322)”, the “Basic Scientific Research Business Fee Project for Universities Directly Under the Inner Mongolia Autonomous Region (BR22-13-02)”, and the “Inner Mongolia Agricultural University Landmark Achievement Project (BZX202211)”.

Institutional Review Board Statement

All animal procedures were conducted according to the “Laboratory Animal Guideline for Ethical Review of Animal Welfare” of the National Standard of the People’s Republic of China (GB/T 35892-2018) [48]. The Animal Welfare and Ethics Committee of Inner Mongolia Agricultural University (NND2024053) approved this study on 5 March 2024.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Acknowledgments

The authors are grateful to Meila Na for assistance in the sample collection and laboratory analysis, and to Renhua Na, Yu Zhang, and Yulan Liu for the experimental design and financial support.

Conflicts of Interest

Author Yulan Liu was employed by the Inner Mongolia Zhamuqin Agriculture and Animal Husbandry Technology Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Abbreviations

The following abbreviations are used in this manuscript:
RDSrumen-degradable starch
NH3-Nammonia nitrogen
BCPbacterial protein
SCFAshort-chain fatty acid
RDPrumen-degradable protein
DMdry matter
CPcrude protein
EEether extract
ADFacid detergent fiber
NDFneutral detergent fiber

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Fermentation 11 00106 g001
Figure 1. Effects of RDS levels on total gas production during in vitro rumen fermentation of alfalfa silage. Data are presented as real-time measurements of total gas produced in mL/h for different groups throughout a 24 h incubation. R1: 14.85% RDS, R2: 16.40% RDS, R3: 18.67% RDS, R4: 20.21% RDS, R5: 21.62% RDS. * means linear: p < 0.05, ** means linear: p < 0.01, + means quadratic: p < 0.05, ++ means quadratic: p < 0.01.
Figure 1. Effects of RDS levels on total gas production during in vitro rumen fermentation of alfalfa silage. Data are presented as real-time measurements of total gas produced in mL/h for different groups throughout a 24 h incubation. R1: 14.85% RDS, R2: 16.40% RDS, R3: 18.67% RDS, R4: 20.21% RDS, R5: 21.62% RDS. * means linear: p < 0.05, ** means linear: p < 0.01, + means quadratic: p < 0.05, ++ means quadratic: p < 0.01.
Fermentation 11 00106 g001
Fermentation 11 00106 g002
Figure 2. The effects of RDS levels on in vitro rumen fermentation Single-factor associative effects indices (SFAEI) and multiple-factors associative effects indices (MFAEI) of alfalfa silage. R1: 14.85% RDS, R2: 16.40% RDS, R3: 18.67% RDS, R4: 20.21% RDS, R5: 21.62% RDS. TSCFA, total short-chain fatty acids; GP, gas production; BCP, bacterial protein.
Figure 2. The effects of RDS levels on in vitro rumen fermentation Single-factor associative effects indices (SFAEI) and multiple-factors associative effects indices (MFAEI) of alfalfa silage. R1: 14.85% RDS, R2: 16.40% RDS, R3: 18.67% RDS, R4: 20.21% RDS, R5: 21.62% RDS. TSCFA, total short-chain fatty acids; GP, gas production; BCP, bacterial protein.
Fermentation 11 00106 g002
Table 1. Chemical compositions of alfalfa forms and RDS.
Table 1. Chemical compositions of alfalfa forms and RDS.
Nutrient 1Corn StalkAlfalfa SilageCornWheatSoybean MealWheat Bran
DM94.4437.4086.9085.7091.2090.2
DE, MJ/kg9.5810.7714.8614.9116.4112.81
CP4.5219.108.5013.5047.6017.4
starch3.54.8070.4063.10--
ADF42.9226.583.604.2010.1013.80
NDF71.3236.679.8012.5019.6040.12
EE3.353.503.841.987.124.39
RDS--38.0849.71--
RDP-58.00----
1 DM: dry matter, DE: digestible energy, CP: crude protein, ADF: acid detergent fiber, NDF: neutral detergent fiber, EE: ether extract, RDS: rumen-degradable starch, RDP: rumen-degradable protein.
Table 2. Composition and nutritional level of substrate in the in vitro experiment (air-dry basis).
Table 2. Composition and nutritional level of substrate in the in vitro experiment (air-dry basis).
IngredientsTreatment 1Nutrient 3Treatment
R1R2R3R4R5R1R2R3R4R5
Corn stalk1010101010DM66.7466.1365.4365.2765.12
Alfalfa silage4040404040DE, MJ/kg12.0312.0712.0812.0711.98
Wheat010233343.5CP14.6414.6714.4514.5614.73
Corn393019100starch28.5528.4728.8528.8028.37
Soybean meal54210ADF21.2721.2021.0821.0721.12
Wheat bran43.5333.5NDF30.2230.1930.1430.3230.65
Soybean oil00.5111EE4.054.254.304.043.78
Calcium Hydrogen Phosohate0.250.250.250.250.25RDS14.8516.4018.6720.2121.62
Limestone0.750.750.750.750.75RDP6.326.326.326.326.32
Salt0.50.50.50.50.5RDS/RDP2.352.602.953.203.47
Premix 20.50.50.50.50.5
1 R1: 14.85% RDS, R2: 16.40% RDS, R3: 18.67% RDS, R4: 20.21% RDS, R5: 21.62% RDS. 2 The premix contained per kg of diet: vitamin A 6000 IU, vitamin D3 2000 IU, vitamin E 15 IU, vitamin K3 1.8 mg, vitamin B1 0.35 mg, vitamin B2 8.5 mg, vitamin B6 0.9 mg, vitamin B12 0.03 mg, D-pantothenic acid 16 mg, nicotinic acid 22 mg, folic acid 1.5 mg, biotin 0.15 mg, Cu 8 g, Fe 40 mg, Mn 20 mg, Zn 40 mg, I 0.8 mg, Se 0.3 mg, Co 0.3 mg. 3 DM: dry matter, DE: digestible energy, CP: crude protein, ADF: acid detergent fiber, NDF: neutral detergent fiber, EE: ether extract, RDS: rumen-degradable starch, RDP: rumen-degradable protein.
Table 3. The effects of RDS levels on in vitro rumen fermentation parameters of alfalfa silage.
Table 3. The effects of RDS levels on in vitro rumen fermentation parameters of alfalfa silage.
ItemsGroups 1SEMp-Values 2
R1R2R3R4R5RDSLQ
3 h
pH6.676.656.656.636.640.010.2780.0350.101
NH3-N, mg/100 mL21.8321.9420.6420.3821.440.370.2850.0600.082
BCP, mg/100 mL36.34 c35.12 c38.44 bc42.98 a40.33 ab0.690.0040.0030.017
α-amylase, U/dL272.39 c302.15 b319.89 ab317.60 ab343.35 a26.60.0020.0010.001
6 h
pH6.466.406.416.376.340.030.4620.0630.191
NH3-N, mg/100 mL23.2123.0022.3620.9221.780.440.1770.0300.097
BCP, mg/100 mL40.0840.0541.7643.8241.450.640.8600.3580.662
α-amylase, U/dL256.16 c275.42 bc295.26 ab299.93 a286.51 ab37.080.0050.0050.001
12 h
pH6.186.146.136.096.090.030.6320.0990.263
NH3-N, mg/100 mL26.1926.5525.1024.2523.770.670.4120.0470.141
BCP, mg/100 mL47.8547.6448.6050.5051.970.370.1620.0130.296
α-amylase, U/dL203.72235.77262.66274.68244.3515.870.1710.0680.038
24 h
pH6.046.005.985.946.010.040.8130.4960.055
NH3-N, mg/100 mL31.8031.7630.9031.4031.670.450.7730.6700.576
BCP, mg/100 mL55.5757.1355.7654.1854.890.370.7570.3760.635
α-amylase, U/dL187.89179.14195.48240.41211.8230.380.0750.0430.141
1 R1: 14.85% RDS, R2: 16.40% RDS, R3: 18.67% RDS, R4: 20.21% RDS, R5: 21.62% RDS. NH3-N: ammonia nitrogen; BCP: bacterial protein. 2 p (RDS) = effect of RDS levels; p (L) = linear; p (Q) = quadratic. a–c: means with different superscripts within the same column differ significantly, p < 0.05.
Table 4. The effects of RDS levels on in vitro rumen fermentation SCFA of alfalfa silage.
Table 4. The effects of RDS levels on in vitro rumen fermentation SCFA of alfalfa silage.
ItemsGroups 1SEMp-Values 2
R1R2R3R4R5RDSLQ
3 h
Acetate12.4712.9812.9913.3612.880.830.9760.6550.815
Propionate4.745.075.175.165.240.310.8440.2640.488
Isobutyrate2.292.202.102.152.000.250.9640.4450.756
Butyrate2.532.472.362.382.170.230.8800.2780.556
Isovaleric4.273.754.024.193.810.430.9240.7530.947
Valerianic2.052.041.911.941.770.190.8770.2790.557
TSCFA28.3528.5128.5429.1827.871.960.9960.9750.960
A/P2.662.562.512.592.440.100.6870.2060.464
6 h
Acetate14.63 c15.21 c15.55 bc16.69 ab17.21 a0.680.0110.0010.001
Propionate5.62 c5.80 c6.05 bc6.48 b7.00 a0.330.0010.0010.001
Isobutyrate2.712.732.692.572.550.110.7570.1970.406
Butyrate2.963.013.023.073.070.140.9860.5470.837
Isovaleric4.474.534.464.524.600.180.9890.6780.897
Valerianic3.353.223.253.263.040.110.4560.1210.270
TSCFA33.7334.5035.0236.5937.471.140.0910.0030.014
A/P2.602.622.572.582.460.080.6590.1880.311
12 h
Acetate23.2624.8724.6224.2425.881.030.5440.1700.405
Propionate9.059.7910.269.9710.240.390.1620.0290.448
Isobutyrate3.333.233.293.432.960.270.8310.5620.697
Butyrate4.694.475.195.005.050.270.3530.1350.328
Isovaleric6.106.006.255.645.640.250.2960.1180.219
Valerianic6.546.427.386.876.440.330.1870.7230.245
TSCFA52.9854.7756.9855.1456.201.340.2870.0960.139
A/P2.572.542.402.432.530.070.4810.3920.250
24 h
Acetate27.0227.6027.4628.0929.161.310.8630.2760.526
Propionate9.6510.2210.3210.7511.090.560.4830.0530.165
Isobutyrate3.824.234.304.624.940.530.6960.1210.314
Butyrate5.805.635.836.666.790.590.5650.2460.116
Isovaleric7.537.397.157.088.500.840.8110.5910.518
Valerianic7.347.447.947.648.970.820.6990.2010.387
TSCFA61.1662.5163.0064.8369.454.020.6960.1510.314
A/P2.802.692.662.612.660.100.8240.2960.461
1 R1: 14.85% RDS, R2: 16.40% RDS, R3: 18.67% RDS, R4: 20.21% RDS, R5: 21.62% RDS. NH3-N: ammonia nitrogen; BCP: bacterial protein. 2 p (RDS) = effect of RDS levels; p (L) = linear; p (Q) = quadratic. a–c: means with different superscripts within the same column differ significantly, p < 0.05.
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Guo, W.; Liu, Y.; Na, M.; Zhang, Y.; Na, R. Effects of Rumen-Degradable Starch Levels on In Vitro Rumen Fermentation and Microbial Protein Synthesis in Alfalfa Silage. Fermentation 2025, 11, 106. https://doi.org/10.3390/fermentation11020106

AMA Style

Guo W, Liu Y, Na M, Zhang Y, Na R. Effects of Rumen-Degradable Starch Levels on In Vitro Rumen Fermentation and Microbial Protein Synthesis in Alfalfa Silage. Fermentation. 2025; 11(2):106. https://doi.org/10.3390/fermentation11020106

Chicago/Turabian Style

Guo, Wenliang, Yulan Liu, Meila Na, Yu Zhang, and Renhua Na. 2025. "Effects of Rumen-Degradable Starch Levels on In Vitro Rumen Fermentation and Microbial Protein Synthesis in Alfalfa Silage" Fermentation 11, no. 2: 106. https://doi.org/10.3390/fermentation11020106

APA Style

Guo, W., Liu, Y., Na, M., Zhang, Y., & Na, R. (2025). Effects of Rumen-Degradable Starch Levels on In Vitro Rumen Fermentation and Microbial Protein Synthesis in Alfalfa Silage. Fermentation, 11(2), 106. https://doi.org/10.3390/fermentation11020106

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