Next Article in Journal
Identification of Simultaneous Occurrence of Amphibian Chytrid Fungi and Ranavirus in South Korea
Previous Article in Journal
Effects of Habitat Differences and Invasive Species Competition on Age and Growth of Triplophysa strauchii
Previous Article in Special Issue
Exploration of Multi-Source Lignocellulose-Degrading Microbial Resources and Bioaugmentation Strategies: Implications for Rumen Efficiency
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Animals
Volume 15
Issue 14
10.3390/ani15142131
animals-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Mixed Ensiling Increases Degradation Without Altering Attached Microbiota Through In Situ Ruminal Incubation Technique

by
Xuanxuan Pu
1,2,3,†,
Min Zhang
4,†,
Jianjun Zhang
4,
Xiumin Zhang
4,*,
Shizhe Zhang
4,
Bo Lin
5,
Tianwei Wang
6,
Zhiliang Tan
4 and
Min Wang
4,*
1
College of Animal Science and Technology, Tarim University, Alar 843300, China
2
Key Laboratory of Tarim Animal Husbandry Science and Technology of Xinjiang Production and Construction Group, Alar 843300, China
3
Key Laboratory of Livestock and Forage Resources Utilization around Tarim, Ministry of Agriculture and Rural Affairs, Alar 843300, China
4
CAS Key Laboratory for Agro-Ecological Processes in Subtropical Region, Hunan Provincial Engineering Research Center for Healthy Livestock and Poultry Production, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha 410125, China
5
College of Animal Science and Technology, Guangxi University, Nanning 530004, China
6
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Animals 2025, 15(14), 2131; https://doi.org/10.3390/ani15142131
Submission received: 19 June 2025 / Revised: 16 July 2025 / Accepted: 16 July 2025 / Published: 18 July 2025
(This article belongs to the Special Issue Maximizing Fiber Utilization for Sustainable, Efficient Ruminant Production)
Browse Figures
Review Reports Versions Notes

Simple Summary

Better utilization of rape straw can provide alternative strategies for the sustainability of ruminants and food production. In the present study, rape straw was mixed ensiled with whole crop corn, and obtained good fermentation quality by enriching lactic acid bacteria. For a better understanding of mixed silage effects on the nutrition and utilization value of rape straw, rape straw was mixed with whole crop corn silage and used as a control group. Results showed that mixed silage destroyed the fiber structure of rape straw, and enhanced in situ nutrient degradation by increasing both rapidly and potentially degradable nutrients. In summary, mixed silage provides a potential strategy to improve nutrient and utilization value of rape straw.

Abstract

Mixed silage can disrupt the girder structure of rape straw, and thus facilitate ruminal degradation. Further investigation is warranted to validate this observation in vivo. The objective of this study was to investigate the degradation kinetics and bacterial colonization of mixed silage during digestion using an in situ ruminal incubation technique. The experiment comprised two treatments: a mixture of rape straw and corn silage (control), and a mixed silage treatment of rape straw and whole crop corn (mixed silage). Three ruminally cannulated Holstein bulls were employed. Substrates were incubated for varying durations (4, 12, 24, 48, 72, 96, 120 and 216 h) to assess substrate degradation kinetics. Bacterial colonization were analyzed after 4- and 48-h incubation time. Mixed ensiling disrupted the fiber structure of rape straw, and thus had lower fiber content compared to the control, as NDF and ADF content ‌decreased by 55 g/kg (678 vs. 623 g/kg) and 27 g/kg (440 vs. 413 g/kg), respectively. Compared to the control group, ruminal DM disappearance of mixed silage significantly (p ≤ 0.05) increased from 315 to 366 g/kg (+16.2%) at an incubation time of 4 h, 552 to 638 g/kg (+15.6%) at 120 h, and 563 to 651 g/kg (+15.6%) at 216 h. Similarly, compared to the control group, NDF disappearance of mixed silage significantly (p ≤ 0.05) rose from 112 to 201 g/kg (+79.5%) at 4 h, 405 to 517 g/kg (+27.7%) at 120 h, and 429 to 532 g/kg (+24.0%) at 216 h. Compared to the control group, soluble and washout nutrient fractions (a) of DM or NDF fraction in mixed silage significantly (p ≤ 0.05) rose from 289 to 340 g/kg (+17.6%), potentially degradable fractions (b) of NDF increased from 310 to 370 g/kg (+19.4%), and the undegraded fraction of NDF (μNDF) decreased from 582 to 471 g/kg (−19.1%). Incubation time, apart from in the mixed ensiling treatment, altered the bacterial community. The study highlights that higher total potentially degradable fractions account for enhanced ruminal substrate degradation of mixed silage.

1. Introduction

About 4.05 million tons of rape straw are produced annually in China, and its disposal has become a concern [1]. Burning, as a traditional way to treat rape straw, causes both environmental pollution and resource waste [1]. Rape straw can be used as a potential feedstuff for ruminants, but the high lignified fiber makes it difficult for ruminants to degrade and utilize [2]. Therefore, strategies are needed to improve the availability of rape straw as ruminant feed. Mixed silage of straw with materials that are rich in water soluble carbohydrates (WSC), such as whole crop corn, promises to exhibit good fermentation quality for long-term storage as a rapid decrease in pH [3,4]. The microorganism enriched during the ensiling process could break down the ligno-cellulosic bonds of the plant cell wall [5,6], which facilitates ruminal microbial colonization and thus enhances the feeding value of straw for ruminants. However, no study has yet been conducted to evaluate effects of mixed silage on the carbohydrate composition of rape straw, which is critical in improving potential utilization of rape straw.
Microorganisms colonizing the rumen play crucial roles in digesting plant materials [7], which can change with prolongation of incubation time [8]. Disparate forages can alter attached microbiota, owing to their chemical composition and ruminal availability differences [9]. For instance, rice straw treated with sodium hydroxide can enrich fibrolytic species compared to untreated rice straw [10]. The hydrolyzed plant cell walls may enhance bacterial colonization by generating substrates that favor specific microorganisms [11]. However, it remains unclear whether changes in the micro-structure of rape straw during the ensiling process could impact ruminal bacterial attachment, and consequently influence ruminal substrate degradation.
We hypothesized that mixed ensiling rape straw with whole crop corn could produce good fermentation quality, and disrupt the fiber structure of rape straw, and thus result in better nutrient and ruminal utilization value of rape straw. This study was designed to investigate fermentation quality in a mixed silage treatment, and observed the fiber structure changes of rape straw during the mixed ensiling process. An in situ ruminal incubation technique was employed to investigate the effects of the mixed silage treatment on ruminal degradation kinetics and the attached bacterial community.

2. Materials and Methods

2.1. Silage Preparation and Sample Collection

Whole crop corn was harvested with a stem cut about 15 cm above the ground level when 50% to 60% of the grain had the characteristics of the dough stage. Rape was harvested 30 d after the end of flowering, and rape straw was air-dried after it was obtained. Both rape straw and whole crop corn were harvested in Hulunbuir City (47°05′ N to 53°20′ N), Inner Mongolia Autonomous Region, China. Prior to ensiling, both rape straw and whole crop corn were chopped into 2–3 cm pieces. For the control treatment, a portion of whole crop corn was packed and sealed into plastic bags (20 cm × 30 cm), and anaerobically fermented for 60 d at room temperature to obtain corn silage, which was then mixed with rape straw at the dry matter (DM) ratio of 1:2 to obtain the mixture of rape straw and corn silage (control). In the preparation of mixed silage, rape straw was combined with whole crop corn at the DM ratio of 2:1. The mixture was sprayed with distilled water to adjust moisture content to around 60%, as described in our previous study [12]. Subsequently, the mixture was packed and sealed into plastic bags (20 cm × 30 cm), and anaerobically fermented for 60 d at room temperature to obtain mixed silage. Both the control and mixed silage were prepared with four replicates. Samples collected for the analysis of fermentation parameters, nutrient compositions and bacterial compositions were according to procedures outlined in our previous studies [12].

2.2. Animals

The experiment was conducted at the beef cattle farm of Guangxi Taiminxing Animal Husbandry development Co., Ltd., Nanning, China. Three ruminally cannulated Holstein bulls (12 mo, 280 ± 7.5 kg) were employed for the in situ incubation. Cattle were offered a high-forage diet consisting of wheat straw and concentrates (Table 1), and fed twice daily at 0800 and 1700 in individual stalls.

2.3. In Situ Ruminal Incubation

Holstein bulls were given 30 d to adapt to the diet and environment before the in situ ruminal incubation. The procedure of incubation with nylon bags in the rumen of animal was slightly modified according to the procedure described by Zhang et al. [14]. Briefly, approximately 6.5 g of substrate (either control or mixed silage) was weighed into polyester bags. For each Holstein bull, a total of 20 bags were prepared for each forage, with 16 bags allocated for degradation kinetics analysis and 4 bags for assessing microbial community changes. Incubation was staggered in time to ensure no more than 12 bags were present in the rumen for each Holstein bull at each time point, ensuring adequate contact between the nylon bags and rumen microbes. For the degradation kinetics analysis, incubation times were set at 4, 12, 24, 48, 72, 96, 120 and 216 h, with duplicate bags collected at each sampling time. For the microbial community analysis, incubation times were set at 4 and 48 h, with duplicate bags collected at each time point. Upon removal from the rumen, bags for degradation kinetics analyses were put into ice water to arrest microbial activity and then dried at 65 °C for the subsequent chemical analysis. Microbial bags were cleaned with phosphate buffer saline (pH = 7.4), and the liquid was gently wrung out. Then, the inner content of the bags was put into a 15-mL tube and stored at −80 °C for microbial DNA extraction.

2.4. Analytical Methods

The DM and crude protein (CP) were determined as outlined in AOAC methods [15]. NDF and ADF were measured as outlined in Van Soest et al. [16], and hemicellulose content was calculated as NDF minus ADF. Scanning electron microscopy (model SU8010, Hitachi, Tokyo, Japan) was employed to obtain images of rape straw according to the manufacturer’s instructions. Lactate was measured according to the method described by Taylor [17]. Individual VFA concentrations were measured using a gas chromatograph (Agilent 7890 A, Agilent Inc., Palo Alto, CA, USA) [18]. The ammonia-N concentration was measured using the phenol–hypochlorite colorimetric method [19].

2.5. DNA Extraction and High-Throughput Sequencing

DNA extraction was performed using methodology modified from that of Ma et al. [20]. Illumina sequencing was performed using the MiSeq platform (Illumina, San Diego, CA, USA) at the Shanghai Biozeron Biological Technology Co., Ltd. (Shanghai, China). After PCR amplification, all amplicon libraries were sequenced and the barcodes and sequencing primers were removed before data processing. Raw reads were processed using SMRT Link Analysis software version 11.0 to obtain demultiplexed circular consensus sequence (CCS) reads with the following settings: minimum number of passes = 3, minimum predicted accuracy = 0.99. Raw reads were processed through SMRT Portal to filter sequences for length (>1000 or <1800 bp) and quality. Sequences were further filtered by removing barcode and primer sequences with Lima Pipeline (Pacific Biosciences demultiplexing barcoded software, https://lima.how/). The circular consensus sequencing reads were clustered into amplicon sequences variants using QIIME 2-data2 [21]. The phylogenetic affiliation of each 16S rRNA gene sequence was analyzed by RDP Classifier (https://mothur.s3.us-east-2.amazonaws.com/wiki/trainset19_072023.rdp.tgz, accessed on 15 July 2025) against the SILVA (SSU138) 16S rRNA database using a confidence threshold of 70% [22].

2.6. Calculations

The exponential model of Ørskov and McDonald [23] was used to analyze degradation kinetics parameters. The effective rumen degradation (ED, g/kg) was calculated according to the method described by McDonald [24]. The undegradable fraction of NDF (μNDF, g/kg) was estimated by 1000 minus NDF disappearance measured at 216-h incubation time.

2.7. Statistical Analysis

Data was analyzed using SPSS 26.0 software (Chicago, IL, USA). Exponential parameters were tested by paired T-tests based on each animal. Nutrient disappearances at each incubation time were analyzed using a linear mixed model with treatment (n = 2) as fixed effect and animal (n = 3) as random effect. A Wilcoxon rank-sum test was employed for analyzing attached bacteria compositions. Principal coordinate analysis (PCoA) using amplicon sequences variants with adonis in vegan of R 4.0.3 based on the Bray–Curtis distance was used to test effects of treatment and incubation time on the attached bacterial community in the substrate. p ≤ 0.05 indicates that a finding was significant, and 0.05 < p ≤ 0.10 indicates a tendency to be significant.

3. Results

Mixed silage exhibited good silage quality, as indicated by the low pH (4.22), ammonia-N (2.66 g/kg), propionate (2.29 g/kg) and butyrate (0.01 g/kg), and good lactate (32.3 g/kg) (Table 2). The mixed silage was dominated by the genuses of Lentilactobacillus (52.6%) and Pediococcus (23.6%) (Table 2).
Compared with the control group, mixed silage had lower NDF (623 vs. 678 g/kg), ADF (413 vs. 440 g/kg) and hemicellulose (210 vs. 238 g/kg) contents, and higher NDS content (377 vs. 322 g/kg) (Table 2). Mixed silage altered the physical structure of rape straw, as indicated by the scanning electron microscopy images (Figure 1). The surface of rape straw was smooth and complete in the control group, while the fiber surface connection in mixed silage was broken, leading to partially regular or irregular pore structures with large numbers of microbes colonized in.
In the present study, mixed silage had distinct kinetics of DM or NDF disappearance compared to those of the control, and exhibited greater (p ≤ 0.05) DM or NDF disappearance at the incubation times of 4, 120 and 216 h than the control (Figure 2).
The control and mixed silage showed distinct in situ kinetics of ruminal disappearance in DM and NDF (Table 3). Mixed silage had greater (p ≤ 0.05) a, a + b, ED2 and ED6 for both in situ DM and NDF disappearance. Mixed silage had greater (p ≤ 0.05) b for in situ NDF disappearance, and tended to have greater b and lower lag for in situ DM disappearance (0.05 < p ≤ 0.10). Mixed silage exhibited greater (p ≤ 0.05) ED and lower (p ≤ 0.05) μNDF than the control group.
The bacterial communities attached to the incubated substrate were greatly affected by the incubation time, independent of the treatment. Mixed silage had a similar relative abundance of bacteria at phylum and genus levels (p > 0.10), when compared with the control group (Table 4). Extending incubation time from 4 to 48 h enriched the phyla Proteobacteria, Fibrobacteres and Actinobacteria, and genuses Kineothrix, Lacrimispora, Negativibacillus, Stenotrophomonas and Fibrobacter (p ≤ 0.05), and decreased the phylum Bacterodietes, and genuses Prevotella and Paraprevotella (p ≤ 0.05) (Table 4). The PCoA analyses showed that incubation time, rather than treatment, led to the distinct clustering, with PC 1 and PC 2 explaining 30% and 23% of variation in bacterial amplicon sequences variants, respectively (Figure 3, PIncubation time ≤ 0.05).

4. Discussion

Farmers quickly evaluate the quality of silage by its appearance, color, smell, water content and pH [25,26]. It has been reported that a suitable water content of silage can reduce the likelihood of bad fermentation, and that silage ferments well when the pH is below 4.2 [4,27]. Lactic acid bacteria in silage can use carbohydrates to produce lactate, which reduces pH value and inhibits the multiplication of other microorganisms [28,29]. Butyrate is a product of poorly fermented silage and carries the risk of causing clinical ketosis in livestock, and was present at a low level as 0.01 g/kg in the product of mixed silage. The pH, desirable lactate content and low butyrate content indicated that the mixed silage we produced was of good quality.
Enrichment of homofermentative Pediococcus [30] and heterofermentative Lentilactobacillus [31,32] contributed to the favorable fermentative quality in the mixed silage. Erwinia, Pantoea and Stenotrophomonas are widely distributed in soil and plants [33,34,35], and their appearance in the mixed silage might be due to the production process of rape straw. In summary, mixed silage exhibited good fermentation quality by enriching Lentilactobacillus and Pediococcus.
Silage could effectively reduce the fiber content in crop straw. Yang et al. [36] have reported that the NDF and ADF content in Leymus chinensis is decreased after ensiling. A previous study showed that silage treatment decomposed the platelet structure of epicuticular wax crystals on the leaves of fresh alfalfa [37], so the fermentation of microorganisms in the mixed silage could impact on the micro-structure of forage. Similarly, the mixed silage treatment reduced the NDF and ADF content of crop straw in the present study, as the surface of rape straw was destroyed. This change comes from the microorganisms that decompose and ferment substances in straw to obtain energy and nutrients during the ensiling process [38].
Disruption of fiber structure helps to partially hydrolyze hemicellulose and solubilize lignin, leading to an increased surface for the colonization of ruminal microorganisms [39], and thus facilitates rumen microbial degradation, especially for complicated plant cell carbohydrates [40,41], which is important for the utilization of straw [38]. That is also the reason for the greater ruminal degradation of mixed silage in the present study. Such enhanced ruminal microbial degradation can be attributed to the destruction of rape straw during the ensiling process. In future, an in vivo study will be conducted to further analyze the effects of silage feeding on production parameters.
Mixed silage had greater a for both DM and NDF disappearance in our study. The greater a in the mixed silage could be attributed to the greater organic acids and monosaccharides contents. Similar findings were reported by Thomas et al. [42], who observed that the greater washout fractions in sorghum silage are associated with higher water-soluble carbohydrate concentrations. Furthermore, the damage to the surface of rape straw during the ensiling process may lead to the detachment of minute fragments, allowing them to pass through the aperture of the polyester bags, leading to greater rapidly soluble and washout nutrient fractions in the mixed silage. Mixed silage enhanced ruminal NDF availability in the present study, which was consistent with findings observed by Beauchemin et al. [43]. However, some studies have suggested that the reduction in structure carbohydrate content of straw may lead to decreased fiber disappearance [44]. Although straw treatments can increase the fraction of the more readily available portion of the NDF [45], they may also result in a greater proportion of undegradable fiber in NDF residue [46]. In the current study, mixed ensiling demonstrated the ability to reduce undegradable fiber in rape straw, as evidenced by the increased a + b and ED.
Bacteria play important roles in the carbohydrate degradation and utilization of straw by attaching to the surface [41,47]. The change of remaining biodegradable nutrients in the nylon bags due to pasture degradation will select the microorganism attached to it [9]. In our study, the mixed silage treatment scarcely altered the bacteria composition attached to the incubated substrate. Pu et al. [48] also reported that the bacterial community attached was not affected by the type of straw (rice or wheat straw). However, Liu et al. [9] reported that the microbial community attached was strongly affected by the straw type (rice straw or alfalfa hay). These different results may be partly explained by differences in the content of chemical ingredients and availability between the straws. The enhanced rumen degradation observed for mixed silage may be attributed to easier access of carbohydrate enzymes resulting from the disruption of rape straw during the ensiling process, rather than relying on the bacterial attachment. Incubation time was another factor affecting ruminal microbiota. The phylum Fibrobacteres is adept at degrading plant cell walls [49], facilitating the degradation of intricate crystalline regions of straw in later incubation stages [41,47]. Both the phyla Bacteroidetes and the genus Prevotella are adept at colonizing and utilizing soluble carbohydrates in the early incubation period [8]. That is the reason for the enrichment of the phyla Bacteroidetes and genus Prevotella in the early incubation period, and the phyla Fibrobacteres in the later incubation time in the present study. Furthermore, 48 h of incubation time also enriched the genuses Stenotrophomonas, Papillibacter, Kineothrix, Lacrimispora and Negativibacillus, which might also contribute to the degradation of structural carbohydrate and warrant further investigation.

5. Conclusions

Mixed silage disrupts the fiber structure of rape straw, resulting in higher NDS content and lower fiber content in the mixed silage. This treatment enhances in situ ruminal DM and NDF degradation by a creating higher total potentially degradable fractions in the mixed silage. Such enhanced rumen degradation is not associated with changes in attached bacterial communities. Instead, incubation time had a great influence on the bacterial communities, enriching the phyla Fibrobacteres and the genuses Stenotrophomonas, Papillibacter, Kineothrix, Lacrimispora and Negativibacillus at 48 h of incubation. In summary, the mixed silage treatment improve the feed utilization of rape straw by creating easier physical accessibility.

Author Contributions

Conceptualization, X.Z. and M.W.; methodology, X.P., M.Z., X.Z. and M.W.; validation, J.Z., S.Z., B.L., T.W. and Z.T.; formal analysis, X.P. and M.Z.; investigation, X.P. and M.Z.; resources, B.L. and T.W.; data curation, M.Z., X.P., J.Z. and S.Z.; writing—original draft, X.P. and M.Z.; writing—review and editing, X.Z. and M.W.; supervision, X.Z., M.W. and Z.T.; project administration, M.W.; funding acquisition, X.P., M.W. and X.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Hunan Province Science and Technology Plan (2022NK2021), The Science and Technology Innovation Program of Hunan Province (2023RC3206), the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No. XDA26040203), the National Key R&D Program of China (2021YFD1301003, 2023YFD1300900), and the President’s Fund of Tarim University (TDZKBS202518).

Institutional Review Board Statement

Animal related procedures were approved by the animal care committee (approval number PA20250326019), College of Animal Science and Technology, Tarim University, Alar, Xinjiang, China.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available upon request from the corresponding author.

Acknowledgments

We gratefully acknowledge the provision of materials needed in our experiment by Yiming Cheng.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Gaballah, E.S.; Abomohra, A.E.; Xu, C.; Elsayed, M.; Abdelkader, T.K.; Lin, J.; Yuan, Q. Enhancement of biogas production from rape straw using different co-pretreatment techniques and anaerobic co-digestion with cattle manure. Bioresour. Technol. 2020, 309, 123311. [Google Scholar] [CrossRef] [PubMed]
  2. Griffith, C.; Ribeiro, G.O.; Oba, M.; McAllister, T.A.; Beauchemin, K.A. Potential for improving fiber digestion in the rumen of cattle (Bos taurus) through microbial inoculation from bison (Bison bison): In situ fiber degradation. J. Anim. Sci. 2017, 95, 2156–2167. [Google Scholar] [CrossRef] [PubMed]
  3. Zhang, Q.; Zhao, M.; Wang, X.; Yu, Z.; Na, R. Ensiling alfalfa with whole crop corn improves the silage quality and in vitro digestibility of the silage mixtures. Grassl. Sci. 2017, 63, 211–217. [Google Scholar] [CrossRef]
  4. Zhao, C.; Wang, L.; Ma, G.; Jiang, X.; Yang, J.; Lv, J.; Zhang, Y. Cellulase Interacts with Lactate Bacteria to Affect Fermentation Quality, Microbial Community, and Ruminal Degradability in Mixed Silage of Soybean Residue and Corn Stover. Animals 2021, 11, 334. [Google Scholar] [CrossRef] [PubMed]
  5. Xing, L. Study on the Effects of Lactobacillus and Cellulase Additives on the Quality of Different Silage. Master’s Thesis, China Agricultural University, Beijing, China, 2004. [Google Scholar]
  6. Jiang, B.W. Effects of Enzyme Bacteria Mixed Treatment of Roughage on Fiber Structure, Growth Performance and Rumen Microflora of Tan Sheep. Ph.D. Thesis, Ningxia University, Ningxia, China, 2021. [Google Scholar]
  7. Mizrahi, I.; Wallace, R.J.; Moraïs, S. The rumen microbiome: Balancing food security and environmental impacts. Nat. Rev. Microbiol. 2021, 19, 553–566. [Google Scholar] [CrossRef] [PubMed]
  8. Cheng, Y.; Wang, Y.; Li, Y.; Zhang, Y.; Liu, T.; Wang, Y.; Sharpton, T.J.; Zhu, W. Progressive colonization of bacteria and disappearance of rice straw in the rumen by Illumina Sequencing. Front. Microbiol. 2017, 8, 2165. [Google Scholar] [CrossRef] [PubMed]
  9. Liu, J.; Zhang, M.; Xue, C.; Zhu, W.; Mao, S. Characterization and comparison of the temporal dynamics of ruminal bacterial microbiota colonizing rice straw and alfalfa hay within ruminants. J. Dairy Sci. 2016, 99, 9668–9681. [Google Scholar] [CrossRef] [PubMed]
  10. Koike, S.; Yabuki, H.; Kobayashi, Y. Interaction of rumen bacteria as assumed by colonization patterns on untreated and alkali-treated rice straw. J. Anim. Sci. 2014, 85, 524–531. [Google Scholar] [CrossRef]
  11. Meale, S.J.; Beauchemin, K.A.; Hristov, A.N.; Chaves, A.V.; McAllister, T.A. Board-invited review: Opportunities and challenges in using exogenous enzymes to improve ruminant production. J. Anim. Sci. 2014, 92, 427–442. [Google Scholar] [CrossRef] [PubMed]
  12. Pu, X.X.; Zhang, X.M.; Yi, S.Y.; Wang, R.; Li, Q.S.; Zhang, W.Q.; Qu, J.J.; Huo, J.B.; Lin, B.; Tan, B.; et al. Mixed ensiling plus nitrate destroy fiber structure of rape straw, increase degradation and reduce methanogenesis through in vitro ruminal fermentation. J. Sci. Food Agric. 2024, 104, 3428–3436. [Google Scholar] [CrossRef] [PubMed]
  13. NY/T 815-2004; Beef Cattle Feeding Standards. The Standardization Administration of the People’s Republic of China: Beijing, China, 2004.
  14. Zhang, X.M.; Gruninger, R.J.; Alemu, A.W.; Wang, M.; Tan, Z.L.; Kindermann, M.; Beauchemin, K.A. 3-Nitrooxypropanol supplementation had little effect on fiber degradation and microbial colonization of forage particles when evaluated using the in situ ruminal incubation technique. J. Dairy Sci. 2020, 103, 8986–8997. [Google Scholar] [CrossRef] [PubMed]
  15. Association of Official Analytical Chemists (AOAC). Official Methods of Analysis, 18th ed.; AOAC International: Gaithersburg, MD, USA, 2005. [Google Scholar]
  16. Van Soest, P.J.; Robertson, J.B.; Lewis, B.A. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci. 1991, 74, 3583–3597. [Google Scholar] [CrossRef] [PubMed]
  17. Taylor, K.A.C.C. A simple colorimetric assay for muramic acid and lactic acid. Appl. Biochem. Biotech. 1996, 56, 49–58. [Google Scholar] [CrossRef]
  18. Wang, M.; Sun, X.Z.; Janssen, P.H.; Tang, S.X.; Tan, Z.L. Responses of methane production and fermentation pathways to the increased dissolved hydrogen concentration generated by eight substrates in in vitro ruminal cultures. Anim. Feed. Sci. Technol. 2014, 194, 1–11. [Google Scholar] [CrossRef]
  19. Weatherburn, M. Phenol-hypochlorite reaction for determination of ammonia. Anal. Chem. 1967, 39, 971–974. [Google Scholar] [CrossRef]
  20. Ma, Z.; Wang, R.; Wang, M.; Zhang, X.; Mao, H.; Tan, Z. Short communication: Variability in fermentation end-products and methanogen communities in different rumen sites of dairy cows. J. Dairy Sci. 2018, 101, 5153–5158. [Google Scholar] [CrossRef] [PubMed]
  21. Callahan, B.J.; McMurdie, P.J.; Rosen, M.J.; Han, A.W.; Johnson, A.J.; Holmes, S.P. DADA2: High-resolution sample inference from Illumina amplicon data. Nat. Methods 2016, 13, 581–583. [Google Scholar] [CrossRef] [PubMed]
  22. Amato, K.R.; Yeoman, C.J.; Kent, A.; Righini, N.; Carbonero, F.; Estrada, A.; Gaskins, H.R.; Stumpf, R.M.; Yildirim, S.; Torralba, M.; et al. Habitat degradation impacts black howler monkey (Alouatta pigra) gastrointestinal microbiomes. J. ISME 2013, 7, 1344–1353. [Google Scholar] [CrossRef] [PubMed]
  23. Ørskov, E.; McDonald, I. The estimation of protein degradability in the rumen from incubation measurements weighted according to rate of passage. J. Agric. Sci. 1979, 92, 499–503. [Google Scholar] [CrossRef]
  24. McDonald, I. A revised model for the estimation of protein degradability in the rumen. J. Agric. Sci. 1981, 96, 251–252. [Google Scholar] [CrossRef]
  25. Borreani, G.; Tabacco, E.; Schmidt, R.J.; Holmes, B.J.; Muck, R.E. Silage review: Factors affecting dry matter and quality losses in silages. J. Dairy Sci. 2018, 101, 3952–3979. [Google Scholar] [CrossRef] [PubMed]
  26. Carvalho, B.F.; Sales, G.F.C.; Schwan, R.F.; Ávila, C.L.S. Criteria for lactic acid bacteria screening to enhance silage quality. J. Appl. Microbiol. 2021, 130, 341–355. [Google Scholar] [CrossRef] [PubMed]
  27. Ni, K.; Wang, F.; Zhu, B.; Yang, J.; Zhou, G.; Pan, Y.; Tao, Y.; Zhong, J. Effects of Lactate bacteria and molasses additives on the microbial community and fermentation quality of soybean silage. Bioresour. Technol. 2017, 238, 706–715. [Google Scholar] [CrossRef] [PubMed]
  28. Bonaldi, D.S.; Carvalho, B.F.; Ávila, C.L.D.S.; Silva, C.F. Effects of Bacillus subtilis and its metabolites on corn silage quality. Lett. Appl. Microbiol. 2021, 73, 46–53. [Google Scholar] [CrossRef] [PubMed]
  29. Su, R.; Ni, K.; Wang, T.; Yang, X.; Zhang, J.; Liu, Y.; Shi, W.; Yan, L.; Jie, C.; Zhong, J. Effects of ferulic acid esterase-producing Lactobacillus fermentum and cellulase additives on the fermentation quality and microbial community of alfalfa silage. PeerJ 2019, 7, e7712. [Google Scholar] [CrossRef] [PubMed]
  30. Dong, Z.; Shao, T.; Li, J.; Yang, L.; Yuan, X. Effect of alfalfa microbiota on fermentation quality and bacterial community succession in fresh or sterile Napier grass silages. J. Dairy Sci. 2020, 103, 4288–4301. [Google Scholar] [CrossRef] [PubMed]
  31. Muck, R.E.; Nadeau, E.M.G.; McAllister, T.A.; Contreras-Govea, F.E.; Santos, M.C.; Kung, L., Jr. Silage review: Recent advances and future uses of silage additives. J. Dairy Sci. 2018, 101, 3980–4000. [Google Scholar] [CrossRef] [PubMed]
  32. Wu, B.; Hu, Z.; Wei, M.; Yong, M.; Niu, H. Effects of inoculation of Lactiplantibacillus plantarum and Lentilactobacillus buchneri on fermentation quality, aerobic stability, and microbial community dynamics of wilted Leymus chinensis silage. Front. Microbiol. 2022, 13, 928731. [Google Scholar] [CrossRef] [PubMed]
  33. Dutkiewicz, J.; Mackiewicz, B.; Lemieszek, M.K.; Golec, M.; Milanowski, J. Pantoea agglomerans: A mysterious bacterium of evil and good. Part IV beneficial effects. Ann. Agric. Environ. Med. 2016, 23, 206–222. [Google Scholar] [CrossRef] [PubMed]
  34. Yao, B.; Huang, R.; Zhang, Z.; Shi, S. Seed-Borne Erwinia persicina Affects the Growth and Physiology of Alfalfa (Medicago sativa L.). Front. Microbiol. 2022, 13, 891188. [Google Scholar] [CrossRef] [PubMed]
  35. Kaparullina, E.; Doronina, N.; Chistyakova, T.; Trotsenko, Y. Stenotrophomonas chelatiphaga sp. nov., a new aerobic EDTA-degrading bacterium. Syst. Appl. Microbiol. 2009, 32, 157–162. [Google Scholar] [CrossRef] [PubMed]
  36. Yang, H.; Zhang, Q.; Hou, J.; Yu, Z. Effects of biological additives on ultrastructure and fiber content of Leymus chinensis silage. Acta Pratacult. Sin. 2016, 25, 94–101. [Google Scholar]
  37. Hu, Z.; Niu, H.; Tong, Q.; Chang, J.; Yu, J.; Li, S.; Zhang, S.; Ma, D. The Microbiota Dynamics of Alfalfa Silage During Ensiling and After Air Exposure, and the Metabolomics After Air Exposure Are Affected by Lactobacillus casei and Cellulase Addition. Front. Microbiol. 2020, 11, 519121. [Google Scholar] [CrossRef] [PubMed]
  38. Ávila, C.L.S.; Carvalho, B.F. Silage fermentation-updates focusing on the performance of micro-organisms. J. Appl. Microbiol. 2020, 28, 966–984. [Google Scholar] [CrossRef] [PubMed]
  39. Balan, V.; Bals, B.; Chundawat, S.P.; Marshall, D.; Dale, B.E. Lignocellulosic biomass pretreatment using AFEX. Methods Mol. Biol. 2009, 581, 61–77. [Google Scholar] [CrossRef] [PubMed]
  40. Adesogan, A.T.; Arriola, K.G.; Jiang, Y.; Oyebade, A.; Paula, E.M.; Pech-Cervantes, A.A.; Romero, J.J.; Ferraretto, L.F.; Vyas, D. Symposium review: Technologies for improving fiber utilization. J. Dairy Sci. 2019, 102, 5726–5755. [Google Scholar] [CrossRef] [PubMed]
  41. Moraïs, S.; Mizrahi, I. Islands in the stream: From individual to communal fiber degradation in the rumen ecosystem. FEMS Microbiol. Rev. 2019, 43, 362–379. [Google Scholar] [CrossRef] [PubMed]
  42. Thomas, M.E.; Foster, J.L.; McCuistion, K.C.; Redmon, L.A.; Jessup, R.W. Nutritive value, fermentation characteristics, and in situ disappearance kinetics of sorghum silage treated with inoculants. J. Dairy Sci. 2013, 96, 7120–7131. [Google Scholar] [CrossRef] [PubMed]
  43. Beauchemin, K.A.; Ribeiro, G.O.; Ran, T.; Marami, M.R.; Yang, W.Z.; Khanaki, H.; Gruninger, R.; Tsang, A.; McAllister, T.A. Recombinant fibrolytic feed enzymes and ammonia fibre expansion (AFEX) pretreatment of crop residues to improve fibre degradability in cattle. Anim. Feed. Sci. Technol. 2019, 256, 114260. [Google Scholar] [CrossRef]
  44. Zhang, X.M.; Wang, M.; Yu, Q.; Ma, Z.Y.; Beauchemin, K.A.; Wang, R.; Wen, J.N.; Lukuyu, B.A.; Tan, Z.L. Liquid hot water treatment of rice straw enhances anaerobic degradation and inhibits methane production during in vitro ruminal fermentation. J. Dairy Sci. 2020, 103, 4252–4261. [Google Scholar] [CrossRef] [PubMed]
  45. Hall, M.B.; Pell, A.N.; Chase, L.E. Characteristics of neutral detergent-soluble fiber fermentation by mixed ruminal microbes. Anim. Feed. Sci. Technol. 1988, 70, 23–39. [Google Scholar] [CrossRef]
  46. Robinson, P.H.; Brice, R.E.; Morrison, I.M. Effect of straw treatment and laboratory of incubation on degradation of neutral detergent residue in the rumen. Anim. Feed. Sci. Technol. 1987, 18, 75–82. [Google Scholar] [CrossRef]
  47. Huws, S.A.; Creevey, C.J.; Oyama, L.B.; Mizrahi, I.; Denman, S.E.; Popova, M.; Muñoz-Tamayo, R.; Forano, E.; Waters, S.M.; Hess, M.; et al. Addressing Global Ruminant Agricultural Challenges Through Understanding the Rumen Microbiome: Past, Present, and Future. Front. Microbiol. 2018, 9, 2161. [Google Scholar] [CrossRef] [PubMed]
  48. Pu, X.X.; Zhang, X.M.; Li, Q.S.; Wang, R.; Zhang, M.; Zhang, S.Z.; Lin, B.; Tan, B.; Tan, Z.L.; Wang, M. Comparison of in situ ruminal straw fiber degradation and bacterial community between buffalo and Holstein fed with high-roughage diet. Front. Microbiol. 2023, 13, 1079056. [Google Scholar] [CrossRef] [PubMed]
  49. Sha, Y.; Hu, J.; Shi, B.; Dingkao, R.; Wang, J.; Li, S.; Zhang, W.; Luo, Y.; Liu, X. Characteristics and Functions of the Rumen Microbial Community of Cattle-Yak at Different Ages. Biomed. Res. Int. 2020, 2020, 3482692. [Google Scholar] [CrossRef] [PubMed]
Animals 15 02131 g001
Figure 1. Scanning electron microscopy images (scale bar = 20 μm) of rape straw in treatments without (i.e., control, (A)) or with mixed ensiling (i.e., mixed silage, (B)). Control, a mixture of rape straw and whole crop corn silage; Mixed silage, mixed silage of rape straw and whole crop corn.
Figure 1. Scanning electron microscopy images (scale bar = 20 μm) of rape straw in treatments without (i.e., control, (A)) or with mixed ensiling (i.e., mixed silage, (B)). Control, a mixture of rape straw and whole crop corn silage; Mixed silage, mixed silage of rape straw and whole crop corn.
Animals 15 02131 g001
Animals 15 02131 g002
Figure 2. Effects of mixed silage on the kinetics of in situ ruminal disappearance of DM (A) and NDF (B). Control, a mixture of rape straw and whole crop corn silage; Mixed silage, mixed silage of rape straw and whole crop corn. * indicates significant difference (p ≤ 0.05).
Figure 2. Effects of mixed silage on the kinetics of in situ ruminal disappearance of DM (A) and NDF (B). Control, a mixture of rape straw and whole crop corn silage; Mixed silage, mixed silage of rape straw and whole crop corn. * indicates significant difference (p ≤ 0.05).
Animals 15 02131 g002
Animals 15 02131 g003
Figure 3. Principal coordinate analysis (PCoA) of bacterial community attached to control and mixed silage at 4-h and 48-h incubation. Control, a mixture of rape straw and whole crop corn silage; Mixed silage, mixed silage of rape straw and whole crop corn.
Figure 3. Principal coordinate analysis (PCoA) of bacterial community attached to control and mixed silage at 4-h and 48-h incubation. Control, a mixture of rape straw and whole crop corn silage; Mixed silage, mixed silage of rape straw and whole crop corn.
Animals 15 02131 g003
Table 1. The ingredients and nutrient composition of the cattle diet (g/kg DM).
Table 1. The ingredients and nutrient composition of the cattle diet (g/kg DM).
Item *Value
Ingredients, g/kg
Wheat straw800
Corn115.9
Soybean meal13.3
Cottonseed meal15.2
Wheat bran15.2
Soybean hull11.4
DDGS9.5
NaHCO31.9
NaCl1.9
Premix5.7
Urea10.0
Chemical composition, g/kg
DM921
OM902
CP82
NDF634
ADF377
NEm, MJ/kg4.19
NEf, MJ/kg2.18
* DM, dry matter; OM, organic matter; CP, crude protein; NDF, neutral detergent fiber; ADF, acid detergent fiber; NEm, net energy for maintenance; NEf, net energy for fattening. The premix provided vitamins and microelements per kilogram as follows: 1,000,000 IU of vitamin A; 200,000 IU of vitamin E; 8000 mg of Zn; 80 mg of Se; 120 mg of I; 2000 mg of Fe; 40 mg of Co; 2500 mg of Mn; 2000 mg of Cu. The NEm and NEf were calculated according to Feeding Standard of Beef Cattle (NY/T 815-2004, China [13]).
Table 2. Fermentation parameters and bacterial compositions in the control and mixed silage.
Table 2. Fermentation parameters and bacterial compositions in the control and mixed silage.
Items *ControlMixed Silage
Fermentation end products, g/kg DM
pH-4.22 ± 0.15
Ammonia-N, g/kg DM3.52 ± 0.322.66 ± 0.18
Lactate, g/kg DM20.5 ± 1.2232.3 ± 1.36
Acetate, g/kg DM9.23 ± 1.2324.2 ± 1.56
Propionate, g/kg DM0.26 ± 0.192.29 ± 0.22
Butyrate, g/kg DM0.05 ± 0.020.01 ± 0.01
Nutrient composition, g/kg DM
DM408 ± 1.25405 ± 2.00
CP60.5 ± 0.7861.3 ± 0.89
NDS322 ± 2.12377 ± 1.56
NDF678 ± 2.12623 ± 1.56
ADF440 ± 1.56413 ± 0.98
Hemicellulose238 ± 1.76210 ± 1.35
Bacterial compositions (genus), %
Lentilactobacillus-52.6 ± 2.35
Pediococcus-23.6 ± 2.89
Pantoea-5.29 ± 1.23
Stenotrophomonas-3.23 ± 0.98
Levilactobacillus-2.34 ± 0.78
Erwinia-2.17 ± 1.35
Secundilactobacillus-1.23 ± 0.46
Escherichia-0.82 ± 0.50
Salmonella-0.61 ± 0.23
Sphingobacterium-0.61 ± 0.12
Sarcina-0.53 ± 0.13
others-6.97 ± 2.67
* DM, dry matter; CP, crude protein; NDS, neutral detergent solubles; NDF, neutral detergent fiber; ADF, acid detergent fiber. Control, a mixture of rape straw and whole crop corn silage; Mixed silage, ensiling treatment of rape straw and whole crop corn mixture; -, not measured. The same as below.
Table 3. Effects of mixed ensiling on the kinetics of in situ ruminal disappearance of DM and NDF.
Table 3. Effects of mixed ensiling on the kinetics of in situ ruminal disappearance of DM and NDF.
Items aTreatments bSEMp-Value
ControlMixed Silage
DM
a, g/kg28934012.60.01
b, g/kg27231813.00.08
a + b, g/kg56165922.30.01
c,% h−13.182.370.490.29
Lag, h2.822.460.2320.08
ED2, g/kg44950717.50.02
ED6, g/kg37942716.20.03
NDF
a, g/kg10815912.10.01
b, g/kg31037014.50.04
a + b, g/kg41852926.00.01
c,% h−13.162.510.420.31
lag, h3.132.360.2920.31
ED2, g/kg29336119.40.02
ED6, g/kg21226616.40.04
μNDF, g/kg58247126.00.01
a a soluble and washout nutrient fractions and represents the rapidly degraded component; b, potentially degradable nutrient fractions and represents the slowly degraded component; a + b, total potentially degradable fraction; c, disappearance rate of fraction b; lag, lag time before the initial degradation of fraction b; ED2 and ED6, effective ruminal degradation calculated with passage rate being 0.02/h and 0.06/h respectively; µNDF, undegraded fraction of NDF at 216 h of incubation time. b Control, a mixture of rape straw and whole crop corn silage; Mixed silage, mixed ensiling treatment of rape straw and whole crop corn mixture.
Table 4. Bacterial compositions with relative abundance larger than 0.5% attached to incubated substrate in control or mixed silage.
Table 4. Bacterial compositions with relative abundance larger than 0.5% attached to incubated substrate in control or mixed silage.
Items *TreatmentIncubation Time (h)SEMp-Value
ControlMixed Silage448TreatmentIncubation TimeTreatment × Incubation Time
Phylum Firmicutes43.045.543.545.01.120.330.550.48
Papillibacter2.292.310.983.620.4140.92<0.0010.47
Kineothrix1.461.691.291.860.1510.290.030.06
Lacrimispora1.582.111.062.630.3510.260.010.27
Negativibacillus0.590.540.290.830.1200.700.010.66
Phylum Bacteroidetes32.231.238.724.62.270.59<0.0010.66
Prevotella16.316.823.010.22.360.880.010.93
Paraprevotella5.894.737.123.500.7520.210.010.55
Phylum Proteobacteria16.414.911.320.01.500.23<0.0010.73
Stenotrophomonas9.988.544.5414.01.510.14<0.0010.13
Phylum Fibrobacteres1.792.000.733.060.4590.630.0010.15
Fibrobacter1.751.940.683.010.4580.660.0010.14
Phylum Actinobacteria0.610.490.300.790.0940.370.010.93
* Control, a mixture of rape straw and whole crop corn silage; Mixed silage, mixed silage of rape straw and whole crop corn.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Pu, X.; Zhang, M.; Zhang, J.; Zhang, X.; Zhang, S.; Lin, B.; Wang, T.; Tan, Z.; Wang, M. Mixed Ensiling Increases Degradation Without Altering Attached Microbiota Through In Situ Ruminal Incubation Technique. Animals 2025, 15, 2131. https://doi.org/10.3390/ani15142131

AMA Style

Pu X, Zhang M, Zhang J, Zhang X, Zhang S, Lin B, Wang T, Tan Z, Wang M. Mixed Ensiling Increases Degradation Without Altering Attached Microbiota Through In Situ Ruminal Incubation Technique. Animals. 2025; 15(14):2131. https://doi.org/10.3390/ani15142131

Chicago/Turabian Style

Pu, Xuanxuan, Min Zhang, Jianjun Zhang, Xiumin Zhang, Shizhe Zhang, Bo Lin, Tianwei Wang, Zhiliang Tan, and Min Wang. 2025. "Mixed Ensiling Increases Degradation Without Altering Attached Microbiota Through In Situ Ruminal Incubation Technique" Animals 15, no. 14: 2131. https://doi.org/10.3390/ani15142131

APA Style

Pu, X., Zhang, M., Zhang, J., Zhang, X., Zhang, S., Lin, B., Wang, T., Tan, Z., & Wang, M. (2025). Mixed Ensiling Increases Degradation Without Altering Attached Microbiota Through In Situ Ruminal Incubation Technique. Animals, 15(14), 2131. https://doi.org/10.3390/ani15142131

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop