Assessing the Fermentation Quality, Bacterial Composition and Ruminal Degradability of Caragana korshinskii Ensiled with Oat Grass
by
,
Yao Shen
1,†,
Kun Wang
2,†,
Benhai Xiong
1,
Fuguang Xue
3,
Yajie Kang
1,
Shichao Liu
4 and
Liang Yang
1,* 1
State Key Laboratory of Animal Nutrition and Feeding, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China
2
Department of Animal Science, Shandong Vocational Animal Science and Veterinary College, Weifang 261061, China
3
Nanchang Key Laboratory of Animal Health and Safety Production, Jiangxi Agricultural University, Nanchang 330045, China
4
Weifang Anqiu Ecological Environment Monitoring Center, Weifang 262100, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Fermentation 2025, 11(7), 420; https://doi.org/10.3390/fermentation11070420
Submission received: 6 April 2025
/
Revised: 30 May 2025
/
Accepted: 18 July 2025
/
Published: 20 July 2025
Abstract
The purpose of this study was to explore the effects of co-ensiling Caragana korshinskii with different proportions of oat grass on silage fermentation quality, chemical composition, in situ rumen degradability and in vitro rumen fermentation characteristics. C. korshinskii and oat grass were mixed at different ratios of 100:00, 90:1, 80:2, 70:30, 60:40 and 50:50. Each ratio of mixture was ensiled for 7, 14, 30, 45 and 60 days at room temperature (25 °C), with 30 bags per ratio, for a total of 180 bags. We further investigated the dynamic profiles of the bacterial community during ensiling and in vitro rumen fermentation. The results showed that co-ensiling C. korshinskii and oat grass decreased the pH values and increased the content of lactic acid and acetic acid compared with ensiling C. korshinskii alone. C. korshinskii ensiled with oat grass at a ratio of 70:30 (70% C. korshinskii) showed the best fermentation quality, which was related to higher relative abundance of Lactobacillus and Weissella. The silage with the ratio of 70:30 (70% C. korshinskii) showed higher dry matter digestibility and the more production of gas and total volatile fatty acids, compared with fresh C. korshinskii. In conclusion, C. korshinskii co-ensiled with oat grass at a ratio of 70:30 could enhance the fermentation quality and digestibility of C. korshinskii.
1. Introduction
Ensiling is the most common forage processing and preservation technology used to improve the palatability and digestibility of biomass, based on a spontaneous lactic acid bacteria fermentation under anaerobic conditions [1]. A good fermentation process is the key to producing high quality silage, depending not only on the harvesting and ensiling technique, but also on the type and chemical composition of forage crops [2]. Water-soluble carbohydrates (WSCs) in silage are fermented by epiphytic lactic acid bacteria to lactic acid, which is the fastest and most efficient fermentation acid for dropping silage pH value. Quality silage is produced when lactic acid is the predominant acid. Protein is degraded into amino acids during ensiling and further fermented into ammonia and keto acids by microbial and plant proteases [3]. As an alkali substance, ammonia will inhibit the drop of silage pH values and reduce silage quality. Furthermore, ammonia formation is the result of nitrogen being dissolved in water and therefore cannot be recovered in dry matter (DM), which increases DM loss and reduces feeding value [4]. The sole fermentation of high protein forage (e.g., legume crops) usually results in poor silage quality [5]. Compared to legume crops, cereal crops are rich in WSCs and poor in protein, so ensiling alone or with legume crops could improve silage quality [6,7].
Caragana korshinskii, a perennial leguminous shrub, is widely distributed in arid and semi-arid areas of China as a soil stabilizer to prevent wind erosion and control desertification [8]. Based on our study, the protein content of C. korshinskii is 143.1 g/kg, while its WSC content is 42.5 g/kg DM. As a perennial leguminous shrub, a high protein content and low WSC content of C. korshinskii make it ineffective for fermentation when ensilaging alone. Previous research has shown that co-ensiling it with cereal crops (e.g., ryegrass) improved the fermentation quality of leguminous crops (e.g., Broussonetia papyrifera) [9]. As one of the most important cultivated forages in China, oat grass is productive and rich in WSCs [1]. Therefore, we hypothesized that co-ensiling C. korshinskii with oat grass had the potential to improve the fermentation quality of C. korshinskii. In the present study, we explored the effects of co-ensiling C. korshinskii and different proportions of oat grass on silage fermentation quality, chemical composition, in situ rumen degradability and in vitro rumen fermentation characteristics. We further investigated the dynamic profiles of the bacterial community during ensiling and in vitro rumen fermentation.
2. Materials and Methods
2.1. Harvesting of Fresh Materials and Silage Preparation
Fresh C. korshinskii and oat grass were harvested from a farm in Shangdu County, Ulanqab City, Inner Mongolia (41.03N, 113.10E). Whole plants of oat grass were chopped into 2–3 cm pieces by a forage chopper. Fresh samples of C. korshinskii were kneaded into silk then chopped into fragments similar to oat grass. Fresh C. korshinskii and oat grass were, respectively, sampled for chemical composition analysis. These chopped fresh materials were mixed at different ratios of 100:00 (100% C. korshinskii), 90:10 (90% C. korshinskii), 80:20 (80% C. korshinskii), 70:30 (70% C. korshinskii), 60:40 (60% C. korshinskii) and 50:50 (50% C. korshinskii). For each ratio, 1000 g of the mixture was weighed into vacuum-sealed polyethylene plastic bags (350 mm × 500 mm, Qingdao Lvsheng Biotechnology Co., Ltd., Qingdao, China) and ensiled for 7, 14, 30, 45 and 60 days at room temperature (25 °C), with 30 bags per each ratio, for a total of 180 bags. Half of the six replicates per ratio were randomly selected at 7, 14, 30 and 45 d to determine the bacterial composition of silages.
2.2. Analysis of Chemical Composition and Fermentation Characteristics
Six replicates per ratio for each time point were used to analyze chemical composition and fermentation characteristics. A sample of 200 g collected from each bag was oven-dried at 65 °C for 48 h. The dried samples were then ground to pass through a 1 mm screen for the determination of chemical composition. The DM content was determined by drying the sample to constant weight at 105 °C. The crude protein (CP) content was determined by the Kjeltec™ 8400 analyzer (FOSS, MN, USA). The contents of neutral detergent fiber (NDF) and acid detergent fiber (ADF) were determined using the Ankom 200 fiber analyzer system (Ankom Technology, NY, USA). The content of WSCs was determined using the method described by the Association of Official Analytical Chemists [10]. The ether extract (EE) was repeatedly extracted in the Soxhlet fat extractor, and the filter paper pack containing the sample was taken out from the extraction tube and placed in a constant-temperature oven at 65 °C until a constant weight was achieved. Two grams of the sample were accurately weighed and then carefully moved into the high-temperature furnace to carbonize for the determination of the ash content.
A sample of 20 g samples from each bag was accurately weighed and mixed with 180 mL of distilled water. The mixed solution was further extracted at 4 °C for 24 h, followed by filtration through 4 layers of cheesecloth. The pH value was immediately determined using the SevenGo™ portable pH meter (Mettler Toledo, Greifensee, Switzerland). The content of volatile fatty acids (VFAs) was determined by the Agilent 7890B gas chromatograph (Agilent Technologies, CA, USA) following the method described by Wang et al. [11]. The lactic acid content was determined using a toolkit purchased from Jiancheng Institute of Bioengineering (Nanjing, China). The ammonia-N (NH3-N) content was determined using the phenol-sodium hypochlorite colorimetric method [12].
2.3. Evaluation of Rumen Degradation Characteristics of the Optimized C. korshinskii Silage
Based on the results of chemical composition and fermentation characteristics (Section 2.2), the optimal ratio for co-ensiling C. korshinskii and oat grass was 70:30 (70% C. korshinskii). We further evaluated the rumen degradation of the optimized C. korshinskii silage by the tests of in situ rumen degradability and in vitro rumen fermentation. Three cannulated lactating Holstein cows with an average body weight of 543.3 ± 26.7 kg were selected for the in situ rumen degradability test and in vitro rumen fermentation test. The chemical composition of diet fed for these cows is shown in Supplemental Table S1. The fresh C. korshinskii was applied as the control group, which was compared to the optimized C. korshinskii silage. The in situ rumen degradability test was conducted according to the description by Mehrez and Ørskov [13]. Twenty-eight nylon bags (2 bags per treatment for 7 time points) were placed simultaneously into the rumen of each of three cannulated dairy cows. Two bags per treatment per cow were retrieved after in situ incubation for 1, 2, 4, 8, 12, 24 and 48 h. The bags were rinsed and oven-dried at 65 °C for 48 h. The content of DM was determined as described above, and the disappearance of DM was calculated based on the corresponding amount left in bags after each incubation.
Rumen fluid was collected from three cannulated dairy cows 2 h before morning feeding and filtered through 4 layers of cheesecloth. The buffer solution was prepared according to the method described by Merry et al. [14] and mixed with the filtered rumen fluid in a ratio of 2:1. The in vitro rumen fermentation test was conducted using 120 mL serum bottles, each of which contained 0.5 g (DM) of fresh C. korshinskii sample or the optimized C. korshinskii silage sample and 75 mL of mixed liquid. A total of 42 bottles for two treatments were incubated in a 39 °C constant temperature bath shaker for 1, 2, 4, 8, 12, 24 and 48 h. Three bottles for each treatment were used for the analysis of pH, gas production and VFA at each time point. Samples collected at 4, 12, 24 and 48 h were used for the analysis of the bacterial community composition in fermentation liquid.
2.4. Microbial Community Analysis
Microbial DNA from the silage and fermentation liquid samples were extracted by the E.Z.N.ATMMag-Bind Soil DNA Kit (Omega, GA, USA). The V3-V4 variable region of the 16S rRNA gene was amplified using primers 338F (5′-ACTCCTACGGGAGGCAGCA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). The PCR products were detected by 2% agarose gel electrophoresis and recovered by cutting the gel using the AxyPrepDNA Gel Recovery Kit (AXYGEN). High-throughput sequencing was conducted using the Illumina MiSeq platform (2 × 300 bp) (Major, Shanghai, China). The bioinformatic analysis of the sequence data was performed using the online platform of the Majorbio Cloud Platform (www.majorbio.com).
2.5. Statistical Analysis
The data on silage fermentation characteristics, chemical composition, in situ rumen degradability and in vitro rumen fermentation characteristics were analyzed using the PROC MIXED procedure of SAS 9.4 (SAS Institute, Cary, NC, USA) to conduct one-way analysis of variance and Tukey’s test, as shown in the following model:
where Yi is the dependent variable, μ is the overall mean, Ti is the treatment effect, ei is the residual. Results were presented as means ± sd. A significant difference was considered when p-value < 0.05.
Yi = μ + Ti + ei
3. Results and Discussion
3.1. Chemical Composition of Fresh C. korshinskii and Oat Grass
The chemical composition of fresh C. korshinskii and oat grass is shown in Table 1. The DM content of fresh C. korshinskii was 67.40%, which was much higher than that of the oat grass (20.63%) used in the present study. A high DM content reduces the loss of material effluent and inhibits the growth of Clostridia, which are undesirable microorganisms during ensiling. The CP content of C. korshinskii was 14.31%, which was higher than that of oat grass (11.24%). During the initial ensiling process, one important chemical change is the breakdown of plant proteins, which are first hydrolyzed to amino acids and then to ammonia and amines. The ammonia formed during proteolysis improves the buffer capacity of silages and influences the rapid decrease in silage pH. The low CP content of C. korshinskii favored a rapid decline in silage pH in the present study. The WSC content of C. korshinskii was 4.25%, which was lower than that of oat grass (21.46%). WSCs play an important role in the quality of silage fermentation. Lactic acid bacteria ferment WSCs into lactic acid, which is the most desirable fermentation acid and prompts a rapid decline in silage pH. C. korshinskii ensiled with oat grass increases the content of WSCs and improves the quality of C. korshinskii silage. The NDF and ADF contents of C. korshinskii were 69.97% and 51.14%, respectively, which were higher than those of oat grass (62.40% and 35.24%). The EE and ash contents of C. korshinskii were 3.51% and 4.31%, respectively, which were lower than those of oat grass (3.91% and 6.95%). The chemical composition of fresh C. korshinskii and oat grass is complementary, and C. korshinskii ensiled with oat grass has the potential to produce quality silage.
3.2. The Fermentation Parameters of C. korshinskii Silage
As shown in Table 2, the pH value is one of the main indicators of well-fermented silage, and the optimal pH of silage is commonly considered to be no higher than 4.2. A low pH value is beneficial for preserving silage for a longer time and to keep aerobic stability when feeding [15,16]. In the present study, co-ensiling C. korshinskii and oat grass significantly decreased the pH values of silages (p < 0.001) compared with ensiling C. korshinskii alone. The silage with the ratio of 70:30 (70% C. korshinskii) had the lowest pH value among all ratios throughout the entire silage fermentation process. The change in pH values reflects the metabolism of WSCs and the production of lactic acid. In keeping with the change in pH values, the concentration of lactic acid significantly increased (p < 0.001) when co-ensiling C. korshinskii and oat grass, and the silage with the of 70:30 (70% C. korshinskii) has the highest lactic acid concentration during the whole ensiling process. In the present study, the pH values of C. korshinskii and oat grass silages significantly decreased to about 4.2 during the 14 days of ensiling (p < 0.001), then decreased gradually to about 4.0. The success of the ensiling process is determined during the first two weeks. Quality silages are achieved when sufficient lactic acid is produced, and the pH value rapidly drops to a value between 3.8 and 4.2. The pH value of silage when ensiling C. korshinskii alone was higher than 4.2 during the whole ensiling process. In the present study, the supplementation of oat grass improved the fermentation quality of C. korshinskii silage.
In the present study, co-ensiling C. korshinskii and oat grass significantly increased the acetic acid concentration (p < 0.001) compared with ensiling C. korshinskii alone. In addition, the silage with the of 70:30 (70% C. korshinskii) had the highest acetic acid concentration during the whole ensiling process. The lactic acid bacteria ferment WSCs into lactic acid, and to a lesser extent to acetic acid. In addition, the lactic acid can be converted to acetic acid under some circumstances [17]. The acetic acid and lactic acid are both desirable end products during ensiling process. Butyric acid is commonly produced by Clostridia, which ferment carbohydrates to butyric acid or convert lactic acid to butyric acid [18]. Clostridia are undesirable microorganisms, and they ferment proteins to biogenic amines and can also be transferred to milk through feces and fecal contamination of the udder. In the present study, supplementation of oat grass significantly decreased the pH values of C. korshinskii silages to about 4.2 during the first two weeks of ensiling (p < 0.001). A rapid and sufficient drop in silage pH values inhibits the growth of undesirable microorganisms, such as Clostridia and Enterobacteria. This may be the reason why butyric acid was not detected in the present study.
Ammonia-N is the byproduct of silage fermentation, and it is produced by inherent plant proteolytic enzymes or undesirable bacteria (e.g., Clostridia and Enterobacteria) [18]. The level of NH3-N reflected the degradation of proteins during the ensiling process [19]. Extensive proteolysis, commonly observed in legume silages, does not only cause a reduction of feeding value but also result in the production of toxic compounds (e.g., biogenic amines and branched fatty acids). Moreover, the NH3-N produced by proteolysis improves the buffer capacity of silage, thus counteracting the rapid decrease in silages pH. In the present study, co-ensiling C. korshinskii and oat grass significantly increased the NH3-N content of silages (p < 0.001) compared with ensiling C. korshinskii alone. The silage with the ratio of 70:30 (70% C. korshinskii) had the highest NH3-N content (11.70–22.85 g/kg TN) during the whole ensiling process. Nevertheless, the NH3-N content in our results was lower than that of stylo and alfalfa silage reported by He et al. [20] and lower than that of Broussonetia papyrifera and perennial ryegrass silage reported by Dong et al. [9], but was similar to that of wet sea buckthorn pomace and alfalfa silage reported by Chen et al. [21]. This may be due to the lower CP content of the raw materials used in the present study. In addition, the extent of proteolysis is dependent on the rate and extent of pH decline. The acid environment reduces the activity of plant proteolytic enzymes and inhibits the growth of undesirable bacteria such as Clostridia and Enterobacteria, which can ferment protein to ammonia. A rapid and sufficient drop in pH observed in the present study may be another reason for the low NH3-N content.
3.3. The Chemical Composition of C. korshinskii Silage
As shown in Table 3, increasing the supplementation proportion of oat grass decreased the DM content due to the relatively low DM content of oat grass. The DM content of silage was consistently reduced during ensiling, while the largest reduction occurred in 70% C. korshinskii, followed by 50% C. korshinskii and 60% C. korshinskii. During the ensiling process, desirable and undesirable microorganisms are able to break down WSCs, protein, cellulose and hemicellulose, so the DM content gradually decreases compared with the initial stage of ensiling [22]. The NDF and ADF contents gradually decreased as the ensiling progressed, and a larger reduction was observed in 70% C. korshinskii. The CP content gradually decreased in prolonged ensilage, but the lowest loss occurred in 70% C. korshinskii. As we discussed above, the reduction is due to the degradation of soluble compounds in raw materials by microorganisms. The different change of CP content in 70% C. korshinskii may be due to the higher lactic acid content and the lower pH value, which would inhibit the growth of protein degradation bacteria such as Clostridia and Enterobacteria.
In the present study, C. korshinskii ensiled with oat grass improved the quality of C. korshinskii silage, and the silage with a 70:30 ratio (70% C. korshinskii) showed the best fermentation performance. There were no lactic acid bacteria additives used in the present study. Lactic acid bacteria belong to the epiphytic microorganisms on plant materials, and their population commonly increases substantially during harvesting and ensiling [23]. The best fermentation performance of C. korshinskii silage observed in the 70:30 ratio (70% C. korshinskii) may be due to the suitable content of soluble compounds, which was most favorable for fermentation by epiphytic lactic acid bacteria. Nevertheless, more studies are needed to confirm the relationship between the content of soluble compounds and epiphytic lactic acid bacteria.
3.4. Bacterial Community Profiles During Ensiling
Microorganisms play an important role in the successful outcome of the silage process. Microorganisms can generally be divided into desirable and undesirable microorganisms. The desirable microorganisms are mainly lactic acid bacteria, such as the core genera Lactobacillus, Leuconostoc, Lactococcus, Pediococcus and Streptococcus, as well as the peripheral genera Enterococcus, Sporolactobacillus, Vagococcus and Weissella. All are members of the Bacillota phylum, and all but Sporolactobacillus are members of the Lactobacillales order. In addition, the genus Bifidobacterium (order Bifidobacteriales) also produces lactic acid by fermenting carbohydrates. In general, lactic acid bacteria degrade WSCs into lactic acid, which reduces pH valve and inhibits the growth of undesirable microorganisms. Quality silage is generally achieved when lactic acid is the predominant acid during the ensiling process. Enterobacteria and Clostridia are common undesirable microorganisms, as they not only compete with lactic acid bacteria for WSCs but also degrade protein, causing a reduction in feeding value and producing toxic compounds such as biogenic amines. The dynamic profiles of the bacterial community during the ensiling process at the genus level are shown in Figure 1. In the present study, the average relative abundances of Clostridia and Enterobacteria were less than 1.5% and were not among the top 10 genera. This may be due to the rapid and sufficient drop in silage pH in the present study, inhibiting the activity of Enterobacteria and Clostridia.
Overall, Lactobacillus and Weissella were the main lactic acid bacteria in the present study at any time point throughout the ensiling process (Figure 1). They are among the most commonly observed lactate-producing bacteria in various silages, and their higher abundance usually means more lactic acid production, lower pH values and higher quality silages [24]. Ni et al. [24] reported that Lactobacillus and Weissella were the dominant bacteria in all silage samples and higher relative abundance of these bacteria was observed in high quality silages. Yan et al. [25] found that the addition of lactic acid bacteria increased the relative abundance of Lactobacillus and improved the silage quality of Italian ryegrass. He et al. [20] found that the addition of Moringa oleifera leaf contributed to the improvement of stylo and alfalfa silage and increased the relative abundance of Lactobacillus. In addition, Dong et al. [9] and Chen et al. [21] also reported similar results. In the present study, Lactobacillus and Weissella showed higher relative abundance in 70% C. korshinskii silages than in the other silages. This may explain why the silage with a 70:30 ratio (70% C. korshinskii) showed the best fermentation performance.
3.5. In Situ Rumen Degradability and In Vitro Rumen Fermentation
The in situ rumen degradability of fresh C. korshinskii and C. korshinskii silage at a 70:30 ratio (70% C. korshinskii) is shown in Figure 2a. The in situ rumen DM degradability of C. korshinskii silage at a 70:30 ratio (70% C. korshinskii) was significantly higher than that of fresh C. korshinskii (p < 0.05) at 1, 2, 4, 8, 12, 24 and 48 h post in sacco incubation. After 48 h of incubation, the degradation of DM in C. korshinskii silage at a 70:30 ratio (70% C. korshinskii) increased by 25.92% compared to that of fresh C. korshinskii. Ensiling is the most common feed processing technology based on spontaneous lactic acid bacteria fermentation, which can improve the palatability and digestibility of forage [26]. Cueva et al. [27] reported that ensiling time improved the degradation rate of corn silage, resulting in greater in situ starch disappearance after 150 d of ensiling. During the ensiling process, microorganisms are able to ferment chemical compounds of raw materials, and this process is similar to microbial pre-treatment. This may explain why the DM digestibility of C. korshinskii silage was largely improved.
The pH values of C. korshinskii silage with a 70:30 ratio (70% C. korshinskii) were significantly lower than those of fresh C. korshinskii (p < 0.05) at 1, 2, 4, 8, 12, 24 and 48 h during in vitro rumen fermentation (Figure 2b). The lower pH value of C. korshinskii silage may be due to the higher concentration of total VFAs in the present study. The gas production and total VFA concentration of C. korshinskii silage with a 70:30 ratio (70% C. korshinskii) were significantly higher than those of fresh C. korshinskii (p < 0.05) at 1, 2, 4, 8, 12, 24 and 48 h during in vitro rumen fermentation (Figure 2c, d). The production of gas and total VFAs are associated with the chemical composition and degradability of feed, with readily degradable components producing more gas and total VFAs [28]. The C. korshinskii silage with a 70:30 ratio (70% C. korshinskii) also produced a higher concentration of acetic acid and propionic acid than fresh C. korshinskii (p < 0.05) at 1, 2, 4, 8, 12, 24 and 48 h during in vitro rumen fermentation (Figure 3a,b). The improved performance (i.e., higher DM digestibility and more production of gas and total VFAs) resulted from the in situ rumen degradability and the in vitro rumen fermentation clearly demonstrates that co-ensiling C. korshinskii and oat grass can be used to improve the quality of C. korshinskii as forage for ruminants.
3.6. Bacterial Community Profiles During In Vitro Rumen Fermentation
The dynamic profiles of the bacterial community during in vitro rumen fermentation are shown in Figure 4. Overall, the bacterial community of C. korshinskii silage at a 70:30 ratio (70% C. korshinskii) showed a similar dynamic profile with that of fresh C. korshinskii. The ensiling process just improved the palatability and digestibility of C. korshinskii by the method of microbial pre-treatment, so no significant changes were observed in the bacterial community during in vitro rumen fermentation. It can be inferred that feeding the C. korshinskii silage with a 70:30 ratio (70% C. korshinskii) to ruminants should not have significant negative effects on microbial fermentation in the rumen.
The C. korshinskii silage with a 70:30 ratio (70% C. korshinskii) showed a higher relative abundance of Rikenellaceae_RC9_gut_group compared to fresh C. korshinskii. Rikenellaceae_RC9_gut_group, belonging to the Rikenellaceae family, plays an essential role in crude fiber digestion, producing propionate and acetate as fermentation end products [29]. Qiu et al. [30] found that dietary fiber could stimulate the growth of Rikenellaceae_RC9_gut_group in the gut of broilers. Wei et al. [31] reported that the relative abundance of Rikenellaceae_RC9_gut_group positively correlated with the production of acetic acid and total VFAs. The higher relative abundance of Rikenellaceae_RC9_gut_group observed in the C. korshinskii silage at a 70:30 ratio (70% C. korshinskii) was consistent with the increased production of acetic acid, propionic acid and total VFAs observed in the present study. Our results further demonstrate that C. korshinskii ensiled with oat grass can be used to improve the quality of C. korshinskii as forage for ruminants.
4. Conclusions
In the present study, the silage of C. korshinskii exhibited a lower pH and higher concentrations of lactic acid and acetic acid when co-ensiled with oat grass. The most favorable fermentation quality was achieved when C. korshinskii and oat grass were ensiled at a 70:30 ratio (comprising 70% C. korshinskii). This improved fermentation performance could be linked to the higher relative abundance of Lactobacillus and Weissella in the silage. Additionally, the silage prepared with a 70:30 ratio (70% C. korshinskii) demonstrated enhanced dry matter digestibility and higher gas production, along with increased total volatile fatty acid levels when contrasted with fresh C. korshinskii. In conclusion, co-ensiling Gramineae and Leguminosae can improve the quality of silage.
Supplementary Materials
The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/fermentation11070420/s1, Table S1: The ingredient and chemical composition of total mixed ration fed for cannulated dairy cows.
Author Contributions
Conceptualization, Y.S., K.W. and L.Y.; methodology, Y.S. and K.W.; software, Y.S., F.X. and Y.K.; investigation, B.X. and S.L.; data curation, Y.S. and K.W.; writing—original draft preparation, Y.S. and K.W.; writing—review and editing, Y.S. and K.W.; visualization, Y.S., F.X. and Y.K.; supervision, L.Y.; project administration, L.Y.; funding acquisition, L.Y. All authors have read and agreed to the published version of the manuscript.
Funding
This project is supported by Central Public-interest Scientific Institution Basal Research Fund (No.Y2025YC58), Key R&D Program of Guangdong Province, China (Grant No. 2023B0202140001-3) and Key R&D Program of Shandong Province, China (Grant No. 2022TZXD0016-1).
Institutional Review Board Statement
Not applicable.
Informed Consent Statement
Not applicable.
Data Availability Statement
All the raw sequences were submitted to the NCBI Sequence Read Archive, under accession number SRP699666.
Conflicts of Interest
The authors declare no conflicts of interest.
Abbreviations
| WSCs | Water-soluble carbohydrates |
| DM | Dry matter |
| CP | Crude protein |
| NDF | Neutral detergent fiber |
| ADF | Acid detergent fiber |
| EE | Ether extract |
| VFAs | Volatile fatty acids |
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Figure 1.
The relative abundance of bacterial communities at the genus level in Caragana korshinskii co-ensiled with different proportions of oat grass for 7 (a), 14 (b), 30 (c) and 45 (d) days.
Figure 1.
The relative abundance of bacterial communities at the genus level in Caragana korshinskii co-ensiled with different proportions of oat grass for 7 (a), 14 (b), 30 (c) and 45 (d) days.
Figure 2.
In situ rumen DM degradability (a), pH value (b), gas production (c) and TVFAs (d) of fresh CK and 70:30 silage during in vitro rumen fermentation. Fresh CK, fresh Caragana korshinskii; 70:30, Caragana korshinskii silage with a 70:30 ratio (70% Caragana korshinskii); DM, dry matter; TVFAs, total volatile fatty acids. * p < 0.05.
Figure 2.
In situ rumen DM degradability (a), pH value (b), gas production (c) and TVFAs (d) of fresh CK and 70:30 silage during in vitro rumen fermentation. Fresh CK, fresh Caragana korshinskii; 70:30, Caragana korshinskii silage with a 70:30 ratio (70% Caragana korshinskii); DM, dry matter; TVFAs, total volatile fatty acids. * p < 0.05.
Figure 3.
The concentration of acetic acid (a), propionic acid (b), butyric acid (c), valeric acid (d) and isovaleric acid (e) of fresh CK and 70:30 during in vitro rumen fermentation. Fresh CK, fresh Caragana korshinskii; 70:30, Caragana korshinskii silage with a 70:30 ratio (70% Caragana korshinskii). * p < 0.05.
Figure 3.
The concentration of acetic acid (a), propionic acid (b), butyric acid (c), valeric acid (d) and isovaleric acid (e) of fresh CK and 70:30 during in vitro rumen fermentation. Fresh CK, fresh Caragana korshinskii; 70:30, Caragana korshinskii silage with a 70:30 ratio (70% Caragana korshinskii). * p < 0.05.
Figure 4.
The relative abundance of bacterial communities at the genus level of fresh CK and at a 70:30 ratio during in vitro rumen fermentation for 4, 12, 24 and 48 h. Fresh CK, fresh Caragana korshinskii; 70:30, Caragana korshinskii silage with the ratio of 70:30 (70% Caragana korshinskii).
Figure 4.
The relative abundance of bacterial communities at the genus level of fresh CK and at a 70:30 ratio during in vitro rumen fermentation for 4, 12, 24 and 48 h. Fresh CK, fresh Caragana korshinskii; 70:30, Caragana korshinskii silage with the ratio of 70:30 (70% Caragana korshinskii).
Table 1.
Chemical composition of fresh materials and the count of lactic acid bacteria.
| Items | C. korshinskii | Oat Grass |
|---|---|---|
| Dry matter (% FM 1) | 67.40 ± 1.55 | 20.63 ± 1.03 |
| Crude protein (% DM 2) | 14.31 ± 0.93 | 11.24 ± 0.75 |
| Neutral detergent fiber (% DM) | 69.67 ± 2.78 | 62.40 ± 1.20 |
| Acid detergent fiber (% DM) | 51.14 ± 1.70 | 35.24 ± 1.28 |
| Water-soluble carbohydrate (% DM) | 4.25 ± 0.21 | 21.46 ± 1.16 |
| Ether extraction (% DM) | 3.51 ± 0.13 | 3.91 ± 0.11 |
| Ash (% DM) | 4.31 ± 0.08 | 6.95 ± 0.04 |
| Lactic acid bacteria (Log10 cfu/g FM) | 4.45 ± 0.38 | 4.75 ± 0.36 |
1 FM, fresh matter. 2 DM, dry matter.
Table 2.
Effects of Caragana korshinskii co-ensiled with different proportions of oat grass on the fermentation parameters.
Table 2.
Effects of Caragana korshinskii co-ensiled with different proportions of oat grass on the fermentation parameters.
| Item | 100:0 | 90:10 | 80:20 | 70:30 | 60:40 | 50:50 1 | p-Value 2 |
|---|---|---|---|---|---|---|---|
| Ensiled for 7 d | |||||||
| pH | 4.89 ± 0.09 a | 4.38 ± 0.05 b | 4.22 ± 0.06 b | 4.06 ± 0.04 c | 4.40 ± 0.07 b | 4.31 ± 0.13 b | <0.001 |
| NH3-N (g/kg TN 3) | 2.16 ± 0.44 d | 6.08 ± 0.27 c | 6.61 ± 0.15 c | 11.70 ± 0.85 a | 6.37 ± 0.50 c | 7.67 ± 0.68 b | <0.001 |
| Lactic acid (mmol/g) | 6.31 ± 0.16 f | 36.15 ± 1.47 c | 44.82 ± 1.84 b | 49.29 ± 2.03 a | 24.18 ± 1.13 e | 30.53 ± 1.53 d | <0.001 |
| Acetate acid (g/kg DM 4) | 3.33 ± 0.63 d | 8.92 ± 1.05 c | 11.47 ± 0.56 b | 13.92 ± 1.38 a | 11.18 ± 0.71 b | 11.59 ± 1.14 b | <0.001 |
| Ensiled for 14 d | |||||||
| pH | 4.78 ± 0.21 a | 4.21 ± 0.06 b | 4.11 ± 0.03 b | 4.03 ± 0.03 b | 4.23 ± 0.05 b | 4.20 ± 0.04 b | <0.001 |
| NH3-N (g/kg TN) | 2.77 ± 0.36 d | 6.82 ± 0.51 c | 9.85 ± 0.80 b | 13.70 ± 0.56 a | 7.52 ± 0.82 c | 8.76 ± 0.68 b | <0.001 |
| Lactic acid (mmol/g) | 8.30 ± 0.35 d | 53.23 ± 3.64 b | 62.24 ± 3.59 a | 64.30 ± 4.58 a | 45.29 ± 2.97 c | 52.14 ± 2.44 b | <0.001 |
| Acetate acid (g/kg DM) | 4.14 ± 0.49 c | 11.43 ± 0.61 b | 12.63 ± 1.46 b | 18.28 ± 1.74 a | 12.66 ± 1.27 b | 12.85 ± 1.77 b | <0.001 |
| Ensiled for 30 d | |||||||
| pH | 4.53 ± 0.18 a | 4.14 ± 0.03 b | 4.07 ± 0.03 b | 4.02 ± 0.02 b | 4.13 ± 0.06 b | 4.12 ± 0.06 b | <0.001 |
| NH3-N (g/kg TN) | 5.05 ± 0.46 e | 13.46 ± 0.84 c | 16.37 ± 0.38 b | 18.32 ± 0.82 a | 12.05 ± 0.63 d | 13.04 ± 0.53 cd | <0.001 |
| Lactic acid (mmol/g) | 14.29 ± 0.55 d | 57.26 ± 1.73 b | 65.13 ± 2.56 a | 68.12 ± 3.08 a | 50.92 ± 2.21 c | 54.53 ± 2.85 bc | <0.001 |
| Acetate acid (g/kg DM) | 8.36 ± 0.81 c | 16.90 ± 1.94 b | 16.19 ± 1.87 b | 20.29 ± 1.03 a | 13.84 ± 0.49 b | 14.76 ± 1.27 b | <0.001 |
| Ensiled for 45 d | |||||||
| pH | 4.40 ± 0.17 a | 4.13 ± 0.02 b | 4.04 ± 0.04 b | 4.02 ± 0.03 b | 4.12 ± 0.09 b | 4.11 ± 0.07 b | 0.002 |
| NH3-N (g/kg TN) | 5.19 ± 0.42 d | 14.99 ± 0.93 c | 18.88 ± 1.09 b | 21.91 ± 1.35 a | 13.09 ± 1.02 c | 13.75 ± 0.60 c | <0.001 |
| Lactic acid (mmol/g) | 22.25 ± 1.50 d | 60.00 ± 1.52 b | 68.12 ± 3.89 a | 70.73 ± 3.71 a | 53.26 ± 2.47 c | 56.64 ± 1.97 bc | <0.001 |
| Acetate acid (g/kg DM) | 9.32 ± 0.53 d | 17.98 ± 1.71 c | 21.39 ± 1.21 b | 24.84 ± 1.38 a | 16.36 ± 1.32 c | 17.72 ± 1.26 c | <0.001 |
| Ensiled for 60 d | |||||||
| pH | 4.36 ± 0.09 a | 4.09 ± 0.02 b | 4.02 ± 0.03 b | 3.97 ± 0.01 b | 4.06 ± 0.04 b | 4.03 ± 0.09 b | <0.001 |
| NH3-N (g/kg TN) | 5.54 ± 0.27 e | 15.47 ± 0.57 c | 19.31 ± 0.89 b | 22.85 ± 1.37 a | 13.50 ± 0.63 d | 14.92 ± 0.73 cd | <0.001 |
| Lactic acid (mmol/g) | 27.62 ± 1.84 c | 62.91 ± 1.59 b | 70.63 ± 2.78 a | 71.28 ± 1.61 a | 68.41 ± 2.55 a | 71.62 ± 2.43 a | <0.001 |
| Acetate acid (g/kg DM) | 10.11 ± 0.76 d | 20.27 ± 1.75 b | 23.04 ± 1.64 b | 27.18 ± 1.55 a | 19.79 ± 1.51 b | 20.77 ± 1.25 b | <0.001 |
1 Treatment 100:0, 100% Caragana. Korshinskii + 0% oat grass; 90:10, 90% Caragana. korshinskii + 10% oat grass; 80:20, 80% Caragana. korshinskii + 20% oat grass; 70:30, 70% Caragana. korshinskii + 30% oat grass; 60:40, 60% Caragana. korshinskii + 40% oat grass; 50:50, 50% Caragana. korshinskii + 50% oat grass. 2 Tukey’s test was used to compare the means in this study. Different letters following numbers indicate a significant difference (p < 0.05). 3 TN, total nitrogen. 4 DM, dry matter.
Table 3.
Effects of Caragana korshinskii co-ensiled with different proportions of oat grass on chemical composition.
Table 3.
Effects of Caragana korshinskii co-ensiled with different proportions of oat grass on chemical composition.
| Item | 100:0 | 90:10 | 80:20 | 70:30 | 60:40 | 50:50 1 | p-Value 2 |
|---|---|---|---|---|---|---|---|
| Ensiled for 7 d | |||||||
| DM 3 | 66.39 ± 1.62 a | 61.87 ± 0.79 b | 57.37 ± 1.80 c | 52.37 ± 1.89 d | 47.93 ± 1.43 e | 43.27 ± 1.58 f | <0.001 |
| CP | 14.14 ± 0.08 a | 13.96 ± 0.06 a | 13.56 ± 0.10 b | 13.36 ± 0.11 c | 13.01 ± 0.14 d | 12.70 ± 0.13 e | <0.001 |
| NDF | 69.47 ± 0.99 a | 68.30 ± 1.04 ab | 67.97 ± 1.07 ab | 65.18 ± 0.94 b | 66.29 ± 2.15 ab | 65.43 ± 1.72 b | 0.014 |
| ADF | 50.20 ± 1.82 a | 48.88 ± 1.29 ab | 47.04 ± 1.24 b | 43.19 ± 0.74 c | 44.16 ± 1.35 c | 42.82 ± 1.32 c | <0.001 |
| Ensiled for 14 d | |||||||
| DM | 65.52 ± 0.91 a | 60.13 ± 1.34 b | 55.88 ± 1.46 c | 50.23 ± 1.28 d | 46.14 ± 1.26 e | 42.50 ± 1.48 f | <0.001 |
| CP | 14.09 ± 0.09 a | 13.90 ± 0.06 b | 13.49 ± 0.16 c | 13.32 ± 0.10 c | 12.93 ± 0.13 d | 12.64 ± 0.06 e | <0.001 |
| NDF | 68.96 ± 1.79 a | 67.52 ± 1.24 ab | 66.58 ± 0.83 ab | 64.81 ± 0.30 b | 65.83 ± 1.49 b | 65.19 ± 1.32 b | 0.014 |
| ADF | 49.03 ± 1.57 a | 47.06 ± 0.53 ab | 45.92 ± 2.71 ab | 41.06 ± 0.88 c | 43.67 ± 1.42 bc | 41.19 ± 1.84 c | <0.001 |
| Ensiled for 30 d | |||||||
| DM | 63.82 ± 0.93 a | 58.97 ± 0.71 b | 53.90 ± 1.74 c | 48.36 ± 0.99 d | 44.86 ± 1.95 e | 40.40 ± 1.14 f | <0.001 |
| CP | 14.01 ± 0.08 a | 13.85 ± 0.07 ab | 13.43 ± 0.16 abc | 13.29 ± 0.09 abc | 12.88 ± 0.10 bc | 12.59 ± 0.08 c | 0.010 |
| NDF | 67.45 ± 2.50 a | 66.31 ± 1.89 ab | 65.46 ± 1.54 ab | 62.72 ± 0.36 b | 64.78 ± 0.98 ab | 63.02 ± 0.82 b | 0.016 |
| ADF | 47.61 ± 1.05 a | 45.71 ± 1.46 ab | 44.28 ± 0.42 bc | 40.26 ± 0.91 d | 42.16 ± 2.38 cd | 40.39 ± 1.06 d | <0.001 |
| Ensiled for 45 d | |||||||
| DM | 62.75 ± 1.00 a | 57.21 ± 1.23 b | 52.88 ± 2.14 c | 46.29 ± 1.69 d | 43.18 ± 1.09 e | 39.12 ± 1.34 f | <0.001 |
| CP | 13.95 ± 0.07 a | 13.79 ± 0.05 b | 13.38 ± 0.08 c | 13.24 ± 0.10 c | 12.83 ± 0.08 d | 12.54 ± 0.11 e | <0.001 |
| NDF | 66.99 ± 0.16 a | 65.90 ± 1.91 ab | 65.14 ± 0.36 b | 62.18 ± 0.30 c | 64.16 ± 0.32 b | 62.61 ± 0.09 c | <0.001 |
| ADF | 46.46 ± 1.08 a | 44.37 ± 0.64 b | 43.19 ± 1.27 bc | 39.33 ± 0.70 d | 41.59 ± 1.04 c | 39.14 ± 0.76 d | <0.001 |
| Ensiled for 60 d | |||||||
| DM | 62.04 ± 1.06 a | 56.69 ± 0.70 b | 52.07 ± 1.28 c | 45.74 ± 1.44 d | 42.21 ± 1.46 e | 38.06 ± 2.25 f | <0.001 |
| CP | 13.91 ± 0.06 a | 13.73 ± 0.02 b | 13.35 ± 0.07 c | 13.21 ± 0.07 d | 12.79 ± 0.13 e | 12.50 ± 0.05 f | <0.001 |
| NDF | 66.78 ± 0.74 a | 65.36 ± 0.69 ab | 65.02 ± 0.47 ab | 61.16 ± 1.32 d | 63.60 ± 1.67 bc | 61.72 ± 1.18 cd | <0.001 |
| ADF | 46.18 ± 0.28 a | 43.93 ± 1.92 b | 43.01 ± 0.52 b | 38.86 ± 0.73 c | 40.73 ± 1.42 c | 38.65 ± 1.33 c | <0.001 |
1 Treatment 100:0, 100% Caragana. Korshinskii + 0% oat grass; 90:10, 90% Caragana. korshinskii + 10% oat grass; 80:20, 80% Caragana. korshinskii + 20% oat grass; 70:30, 70% Caragana. korshinskii + 30% oat grass; 60:40, 60% Caragana. korshinskii + 40% oat grass; 50:50, 50% Caragana. korshinskii + 50% oat grass. 2 Tukey’s test was used to compare the means in this study. Different letters following numbers indicate a significant difference (p < 0.05). 3 DM, dry matter; CP, crude protein; NDF, neutral detergent fiber; ADF, acid detergent fiber.
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Shen, Y.; Wang, K.; Xiong, B.; Xue, F.; Kang, Y.; Liu, S.; Yang, L. Assessing the Fermentation Quality, Bacterial Composition and Ruminal Degradability of Caragana korshinskii Ensiled with Oat Grass. Fermentation 2025, 11, 420. https://doi.org/10.3390/fermentation11070420
AMA Style
Shen Y, Wang K, Xiong B, Xue F, Kang Y, Liu S, Yang L. Assessing the Fermentation Quality, Bacterial Composition and Ruminal Degradability of Caragana korshinskii Ensiled with Oat Grass. Fermentation. 2025; 11(7):420. https://doi.org/10.3390/fermentation11070420
Chicago/Turabian StyleShen, Yao, Kun Wang, Benhai Xiong, Fuguang Xue, Yajie Kang, Shichao Liu, and Liang Yang. 2025. "Assessing the Fermentation Quality, Bacterial Composition and Ruminal Degradability of Caragana korshinskii Ensiled with Oat Grass" Fermentation 11, no. 7: 420. https://doi.org/10.3390/fermentation11070420
APA StyleShen, Y., Wang, K., Xiong, B., Xue, F., Kang, Y., Liu, S., & Yang, L. (2025). Assessing the Fermentation Quality, Bacterial Composition and Ruminal Degradability of Caragana korshinskii Ensiled with Oat Grass. Fermentation, 11(7), 420. https://doi.org/10.3390/fermentation11070420
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