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12 pages, 1649 KB  
Article
Untargeted GC-MS Metabolic Profiling of Anaerobic Gut Fungi Reveals Putative Terpenoids and Strain-Specific Metabolites
by Lazarina V. Butkovich, Candice L. Swift, Chaevien S. Clendinen, Heather M. Olson, Samuel O. Purvine, Oliver B. Vining and Michelle A. O’Malley
Metabolites 2025, 15(9), 578; https://doi.org/10.3390/metabo15090578 - 29 Aug 2025
Viewed by 836
Abstract
Background/Objectives: Anaerobic gut fungi (Neocallimastigomycota) are biotechnologically relevant, lignocellulose-degrading microbes with under-explored biosynthetic potential for secondary metabolites. Untargeted metabolomic profiling with gas chromatography–mass spectrometry (GC-MS) was applied to two gut fungal strains, Anaeromyces robustus and Caecomyces churrovis, to establish a foundational [...] Read more.
Background/Objectives: Anaerobic gut fungi (Neocallimastigomycota) are biotechnologically relevant, lignocellulose-degrading microbes with under-explored biosynthetic potential for secondary metabolites. Untargeted metabolomic profiling with gas chromatography–mass spectrometry (GC-MS) was applied to two gut fungal strains, Anaeromyces robustus and Caecomyces churrovis, to establish a foundational metabolomic dataset to identify metabolites and provide insights into gut fungal metabolic capabilities. Methods: Gut fungi were cultured anaerobically in rumen-fluid-based media with a soluble substrate (cellobiose), and metabolites were extracted using the Metabolite, Protein, and Lipid Extraction (MPLEx) method, enabling metabolomic and proteomic analysis from the same cell samples. Samples were derivatized and analyzed via GC-MS, followed by compound identification by spectral matching to reference databases, molecular networking, and statistical analyses. Results: Distinct metabolites were identified between A. robustus and C. churrovis, including 2,3-dihydroxyisovaleric acid produced by A. robustus and maltotriitol, maltotriose, and melibiose produced by C. churrovis. C. churrovis may polymerize maltotriose to form an extracellular polysaccharide, like pullulan. GC-MS profiling potentially captured sufficiently volatile products of proteomically detected, putative non-ribosomal peptide synthetases and polyketide synthases of A. robustus and C. churrovis. The triterpene squalene and triterpenoid tetrahymanol were putatively identified in A. robustus and C. churrovis. Their conserved, predicted biosynthetic genes—squalene synthase and squalene tetrahymanol cyclase—were identified in A. robustus, C. churrovis, and other anaerobic gut fungal genera. Conclusions: This study provides a foundational, untargeted metabolomic dataset to unmask gut fungal metabolic pathways and biosynthetic potential and to prioritize future efforts for compound isolation and identification. Full article
(This article belongs to the Section Microbiology and Ecological Metabolomics)
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22 pages, 2868 KB  
Article
Impact of Heat Stress on Rumen Fermentation Patterns and Microbiota Diversity and Its Association with Thermotolerance in Indigenous Goats
by Mullakkalparambil Velayudhan Silpa, Veerasamy Sejian, Chinnasamy Devaraj, Artabandhu Sahoo and Raghavendra Bhatta
Fermentation 2025, 11(8), 450; https://doi.org/10.3390/fermentation11080450 - 1 Aug 2025
Viewed by 703
Abstract
Goats are considered to be the ideal climate-resilient animal species in the tropics. Fewer studies are documented assessing the heat stress response of caprine ruminal microbiota, which can also be a crucial indicator of the resilience and/or adaptability of animals. This study was [...] Read more.
Goats are considered to be the ideal climate-resilient animal species in the tropics. Fewer studies are documented assessing the heat stress response of caprine ruminal microbiota, which can also be a crucial indicator of the resilience and/or adaptability of animals. This study was conducted to comparatively assess the heat stress responses of two indigenous goat breeds, Nandidurga and Bidri, based on changes associated with the rumen fermentation pattern and distribution pattern of rumen microbiota. A total of 24 adult animals were randomly allocated into four groups of six animals each, NC (n = 6; Nandidurga control), NHS (n = 6; Nandidurga heat stress), BC (n = 6; Bidri control) and BHS (n = 6; Bidri heat stress). The animals were reared in climate chambers for a duration of 45 days wherein the NC and BC animals were maintained under thermoneutral temperature while the NHS and BHS animals were subjected to simulated heat stress. Heat stress was observed to significantly reduce the rumen ammonia, extracellular CMCase, intracellular carboxy methyl cellulase (CMCase) and total CMCase both in Nandidurga and Bidri goats. In addition to this, a significant reduction in acetate, propionate and total volatile fatty acids (VFAs) was observed in Nandidurga goats. The V3–V4 16s rRNA sequencing further revealed a significant alteration in the rumen microbiota in heat-stressed Nandidurga and Bidri goats. While both the breeds exhibited nearly similar responses in the rumen microbial abundance levels due to heat stress, breed-specific differences were also observed. Furthermore, the LEFSe analysis revealed a significant alteration in the abundances of microbes at the genus level, which were observed to be relatively greater in Bidri goats than Nandidurga goats. Furthermore, these alterations were predicted to impair the functional pathways, especially pathways associated with metabolism. This study therefore provided an insight into the rumen microbial dynamics in heat-stressed goats. Though both the breeds exhibited excellent resilience to the subjected heat stress, there were relatively less ruminal alterations in Nandidurga goats than in Bidri goats. Full article
(This article belongs to the Special Issue Research Progress of Rumen Fermentation)
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24 pages, 3960 KB  
Article
Effect of Dietary Tyrosine on Behavior and Ruminal Meta-Taxonomic Profile of Altay Sheep with Different Temperaments
by Asmita Thapa, Weidong Huang, Shahab Ur Rehman, Muhammad Hammad Zafar, Jinying Zhang, Luoyang Ding and Mengzhi Wang
Vet. Sci. 2025, 12(8), 684; https://doi.org/10.3390/vetsci12080684 - 22 Jul 2025
Viewed by 470
Abstract
The objective of this study was to evaluate the effect of tyrosine on behavior and ruminal meta-taxonomic profile of Altay sheep. Twelve rams with the lowest behavioral responses to humans and twelve rams with the highest behavioral responses were sorted into calm and [...] Read more.
The objective of this study was to evaluate the effect of tyrosine on behavior and ruminal meta-taxonomic profile of Altay sheep. Twelve rams with the lowest behavioral responses to humans and twelve rams with the highest behavioral responses were sorted into calm and nervous groups, respectively. Following the 2 × 2 factorial design, the rams from each group were equally assigned two treatments of a basal diet and a diet with an extra 4 g of tyrosine for 30 d. The temperament trait, growth performance, rumen fermentation parameters, ruminal epithelium antioxidant capacity, and rumen microbial composition were measured at the end of the feeding experiment. The results showed that tyrosine treatment led to a decreased number of crosses in the arena test in nervous sheep (p < 0.05). The tyrosine treatment led to increased antioxidant markers in the rumen epithelium, such as catalase, total antioxidant capacity, and GSH content in rumen epithelial tissues (p < 0.05) in calm and nervous sheep. The total volatile fatty acids and propionic acid content in the rumen were increased by tyrosine treatment in nervous and calm sheep (p < 0.05). The rumen microbial study revealed that the dominant microbial genera were Cryptobacteroides, Prevotella, Limivicinus, Quinella, UBA1711, RUG740, Sachharofermentans, Limomorpha, Soladiphilus, Flexinia, and others in both the tyrosine treatment and temperament groups. A combined effect of treatment and temperament was seen on the abundance of two microbial genera, UBA1711 and RUG740 (p < 0.05). These findings suggest that tyrosine-treated Altay sheep would experience less stress during production, resulting in reduced oxidative stress in the rumen epithelium and improved propionic acid production in the rumen compared with that of basal-diet-treated Altay sheep. Meanwhile, the effect of the dietary tyrosine treatment on ruminal microbial diversity was lower, suggesting lesser degradation of tyrosine by ruminal microbes in Altay sheep. Full article
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19 pages, 3218 KB  
Article
Multi-Omics Integration Reveals the Impact of Gastrointestinal Microbiota on Feed Efficiency in Tan Sheep
by Guohan Sun, Xiaohong Han, Tonggao Liu, Xinrui Zhang, An Shi, Chong Yang and Jinzhong Tao
Microorganisms 2025, 13(7), 1608; https://doi.org/10.3390/microorganisms13071608 - 8 Jul 2025
Viewed by 579
Abstract
The rumen and intestinal microbiota play a pivotal role in the digestion and absorption processes of ruminants. Elucidating the mechanisms by which gastrointestinal microbiota influence the feed conversion ratio (FCR) in ruminants is significantly important for enhancing feed utilization efficiency in these animals. [...] Read more.
The rumen and intestinal microbiota play a pivotal role in the digestion and absorption processes of ruminants. Elucidating the mechanisms by which gastrointestinal microbiota influence the feed conversion ratio (FCR) in ruminants is significantly important for enhancing feed utilization efficiency in these animals. In this study, RT-qPCR, 16S rRNA sequencing, and metabolomic techniques were systematically employed to compare the microbial community structures in the rumen, cecum, and rectum, as well as the differences in rumen metabolites between high- and low-FCR Tan sheep. The results showed that, compared to the HFCR group of Tan sheep, the LFCR group exhibited a significant reduction in unclassified_f__Selenomonadaceae, Blvii28_wastewater-sludge_group, and Papillibacter in the rumen; a significant increase in Lachnospiraceae_AC2044_group and Sanguibacteroides; a significant reduction in unclassified_f__Peptostreptococcaceae, Clostridium_sensu_stricto_1, and Parasutterella in the cecum; a significant increase in norank_f__Bacteroidales_UCG-001; and a significant reduction in norank_f__Muribaculaceae, Blautia, and Turicibacter in the rectum. There is a significant positive correlation between Parasutterella in the cecum and three microorganisms, including unclassified_f__Selenomonadaceae, in the rumen. Additionally, Blvii28_wastewater-sludge_group was positively correlated with Lactobacillus. Furthermore, unclassified_f__Selenomonadaceae in the rumen was positively correlated with Turicibacter, unclassified_f__Peptostreptococcaceae, and Breznakia in the rectum. Blvii28_wastewater-sludge_group also showed positive correlations with Blautia, norank_f__Muribaculaceae, and Clostridium_sensu_stricto_1, while Papillibacter was positively correlated with Faecalitalea. The metabolomic results indicated that, compared to the HFCR group, 261 differential metabolites, including Phenylacetylglutamine and Populin, in the rumen of Tan sheep in the LFCR group were significantly downregulated, whereas 36 differential metabolites, including Glycyl-L-tyrosine, were significantly upregulated. Furthermore, the rumen microbe unclassified_f__Selenomonadaceae exhibited positive correlations with significantly differential metabolites such as L-tryptophan, Etiocholanolone glucuronide, N-acetyl-O-demethylpuromycin, and 6-deoxyerythronolide B. Blvii28_wastewater-sludge_group and Papillibacter also exhibited positive correlations with Icilin. High and low FCRs in the rumen of Tan sheep were investigated, especially in relation to unclassified_f__Selenomonadaceae, Blvii28_wastewater-sludge_group, and Papillibacter. Correlations can be seen with microorganisms such as Parasutatella and Lactobacillus in the cecum; Turicibacter, norank_f__Bacteroideales_UCG-001, and Blautia in the rectum; and metabolites such as L-tryptophan, Etiocholanolone glucuronide, and N-acetyl-O-demethylpuromycin. This reveals the role of microorganisms in the digestion and absorption of Tan sheep feed, thus providing a preliminary basis for further research on the microbial regulation of ruminant animal feed utilization and a theoretical basis for improving Tan sheep feed utilization efficiency. Full article
(This article belongs to the Special Issue Dietary and Animal Gut Microbiota)
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18 pages, 3150 KB  
Article
Synergistic Adaptations of Yak Rumen Microbiota, Metabolites and Host to Altitudinal
by Jianming Ren, Xiong Ma, Pengfei Zhao, Lan Zhang, Shiyu Tao, Xiangyan Wang and Bingang Shi
Microorganisms 2025, 13(7), 1543; https://doi.org/10.3390/microorganisms13071543 - 30 Jun 2025
Viewed by 517
Abstract
Rumen microbiota and metabolites play important roles in energy metabolism and immune regulation in the host. However, the underlying mechanisms of their interaction with the host to regulate yak plateau adaptation remain unknown. In this study, the effects of altitude on the rumen [...] Read more.
Rumen microbiota and metabolites play important roles in energy metabolism and immune regulation in the host. However, the underlying mechanisms of their interaction with the host to regulate yak plateau adaptation remain unknown. In this study, the effects of altitude on the rumen microbiome, metabolome, and fermentation parameters of yaks were analyzed. The fiber content of pasture grasses increased with altitude, while crude protein content was significantly higher at an altitude of 2800 m (T2800) compared to an altitude of 4500 m (T4500) (p < 0.05). The acetic acid, propionic acid, and volatile fatty acids of yaks in the T4500 group were significantly higher than in the T2800 group (p < 0.05). Simpson’s index of rumen microorganisms in the T4500 group of yaks was significantly higher than in T2800 and T3500 yaks. The relative abundance of Rikenellaceae_RC9_gut_group and Succiniclasticum was significantly higher in T4500 than in T2800, while Prevotella and Streptococcus were more abundant in T2800 than in T4500. Rumen metabolomics analyses revealed that yak rumen metabolites at different altitudes were influenced by forage and altitude, mainly affecting energy metabolism and fatty acid biosynthesis (such as purine and glycerophospholipid metabolism). In summary, altitude may influence rumen microbes and metabolites through pasture nutrient composition. Full article
(This article belongs to the Special Issue Gut Bacterial Community: Competition and Mutualism)
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21 pages, 1321 KB  
Review
Exploration of Multi-Source Lignocellulose-Degrading Microbial Resources and Bioaugmentation Strategies: Implications for Rumen Efficiency
by Xiaokang Lv, Zhanhong Qiao, Chao Chen, Jinling Hua and Chuanshe Zhou
Animals 2025, 15(13), 1920; https://doi.org/10.3390/ani15131920 - 29 Jun 2025
Cited by 1 | Viewed by 523
Abstract
Utilizing straw feed is an effective strategy to optimize straw resource utilization by incorporating microbial degradation agents to expedite lignocellulose breakdown and enhance feed efficiency. Lignocellulose-degrading species and microbial communities are present in various Earth ecosystems, including the rumen of ruminants, insect digestive [...] Read more.
Utilizing straw feed is an effective strategy to optimize straw resource utilization by incorporating microbial degradation agents to expedite lignocellulose breakdown and enhance feed efficiency. Lignocellulose-degrading species and microbial communities are present in various Earth ecosystems, including the rumen of ruminants, insect digestive tracts, forest soil, and microbial populations in papermaking processes. The rumen of ruminants harbors a diverse range of microbial species, making it a promising source of lignocellulose-degrading microorganisms. Exploring alternative systems like insect intestines and forest soil is essential for future research. Current studies primarily rely on traditional microbial isolation techniques to identify lignocellulose-degrading strains, underscoring the necessity to transition to utilizing microbial culturomics and genome-editing technologies for discovering and manipulating cellulose-degrading microbes. This review provides an overview of lignocellulose-degrading microbial communities from diverse environments, encompassing bacterial and fungal populations. It also delves into the use of metagenomic, metatranscriptomic, and metaproteomic approaches to pinpoint highly efficient cellulase genes, along with the application of genome-editing tools for engineering lignocellulose-degrading microorganisms. The primary objective of this review is to offer insights for further exploration of potential lignocellulose-degrading microbial resources and high-performance cellulase genes to enhance roughage utilization in ruminant rumen ecosystems. Full article
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25 pages, 5190 KB  
Article
In Vitro Evaluation of Enzymatically Transformed Alfalfa Saponins on Methane Reduction, Rumen Microbes and Metabolomics in Goats
by Ran Zhang, Xinran Bao, Xingqi Shi, Shixuan Jin, Ying Meng, Zhiwei Li, Zhumei Du and Xuebing Yan
Animals 2025, 15(11), 1516; https://doi.org/10.3390/ani15111516 - 22 May 2025
Cited by 1 | Viewed by 589
Abstract
Alfalfa (Medicago sativa L.) saponins (AS), primarily pentacyclic triterpenoids, may reduce methane emissions from goats (Capra hircus L.). This study evaluated the methane-suppressing potential of Aspergillus niger β-glucosidase-modified AS using in vitro rumen fermentation (0.10 mg/mL inoculum, 24 h incubation, gas [...] Read more.
Alfalfa (Medicago sativa L.) saponins (AS), primarily pentacyclic triterpenoids, may reduce methane emissions from goats (Capra hircus L.). This study evaluated the methane-suppressing potential of Aspergillus niger β-glucosidase-modified AS using in vitro rumen fermentation (0.10 mg/mL inoculum, 24 h incubation, gas chromatography detection). Among the 21 alfalfa cultivars, Pegasis (fall dormancy 9) exhibited the highest antioxidant efficacy (half maximum effective concentration 2.13 mg/mL) and the lowest ferric-reducing activity (0.32 μM Fe2+/g) (p < 0.05). Fresh/silage AS reduced methane proportions to 4.50–5.21% of total gas, while enzymatic biotransformation further decreased it to 3.34–3.48% (p < 0.05). Methanogen abundance declined by 20.10–44.93%, and general anaerobic fungi declined by 34.22–44.66% compared to untreated AS (p < 0.05). Metabolomics linked methane suppression to six pathways, including zeatin biosynthesis (via nucleotide metabolites accumulation) and prolactin signaling pathway (via bioactive molecules downregulation), suggesting impaired methanogen energy metabolism and hydrogen flux redirection as mechanisms. Enzymatic AS also enhanced volatile fatty acid production, indicating improved fiber digestion. These in vitro findings demonstrate that enzyme-treated AS modulates rumen fermentation through dual methane mitigation and nutrient utilization enhancement, offering a sustainable feed additive strategy for livestock. Full article
(This article belongs to the Section Small Ruminants)
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28 pages, 673 KB  
Article
Effects of Chenopodium album L. Substitution Levels and Harvest Time on In Vitro Rumen Fermentation and Methane Production in Early-Fattening Hanwoo Steers
by Narantuya Batburged, Gui-Seck Bae, Gurbazar Damdinsuren, Sang-Yoon Kim, Hye-An Lee, Soo-Yeon Jung, In-Ki Kang, Da-Hyun Choi and Chang-Hyun Kim
Animals 2025, 15(10), 1372; https://doi.org/10.3390/ani15101372 - 9 May 2025
Viewed by 1112
Abstract
This study investigated the feasibility of incorporating Chenopodium album L. (CAL) into ruminant feed ingredients through evaluating the effects of harvest time and substitution levels on in vitro rumen fermentation. In the first phase, a sole-substrate experiment was conducted using CAL harvested from [...] Read more.
This study investigated the feasibility of incorporating Chenopodium album L. (CAL) into ruminant feed ingredients through evaluating the effects of harvest time and substitution levels on in vitro rumen fermentation. In the first phase, a sole-substrate experiment was conducted using CAL harvested from June to August, analyzing its chemical composition and total saponins content. The impact of harvest time on fermentation parameters was assessed with CAL as the sole substrate. The second phase involved a mixed-substrate experiment using an early-fattening Hanwoo diet (30% rice straw and 70% concentrate), where increasing proportions of CAL (control: 0%, T1: 5%, T2: 10%, T3: 15%, and T4: 20%) replaced rice straw. Seasonal variations in CAL composition influenced the fermentation characteristics. CAL harvested in July exhibited higher fermentability, with total volatile fatty acids (TVFAs) reaching 103.87 mM at 72 h. In contrast, CAL harvested in August showed lower fermentability and digestibility. However, August-harvested CAL was selected for the subsequent experiment, as it provided a more practical balance of sufficient biomass yield and a higher saponins concentration, aligned with the study’s methane mitigation objectives, while also exhibiting a fiber composition comparable to that of rice straw. We hypothesized that the saponins in CAL contribute to methane reductions. Supplementation with 15% of CAL significantly reduced methane production per gram of inoculated and digested dry matter (p < 0.05), likely due to differences in crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF), and saponins content. However, despite having the lowest fiber content, T4 (20% CAL) exhibited the lowest in vitro dry matter digestibility (IVDMD), suggesting that factors such as saponins, CAL’s chemical composition, or microbial shifts may have hindered digestibility. Ammonia–nitrogen production increased from 0 to 3 h, but it continuously decreased between 3 and 9 h due to microbial growth and nitrogen assimilation, as microbes incorporate ammonia into their biomass (p < 0.05). Fermentation characteristics further revealed that the acetate-to-propionate (A/P) ratio decreased with increasing CAL levels, with T4 showing the lowest ratio (1.55 at 72 h), confirming a shift toward propionate-based fermentation. Notably, T2 (10% CAL) showed an optimized fermentation efficiency, producing the highest TVFA concentration at 24 h (98.28 mM). These findings highlight the potential for using CAL as a functional feed ingredient, with moderate substitution levels (10–15%) enhancing fermentation efficiency while reducing methane production. Full article
(This article belongs to the Section Animal Nutrition)
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30 pages, 13959 KB  
Article
A Comparison Between High- and Low-Performing Lambs and Their Impact on the Meat Quality and Development Level Using a Multi-Omics Analysis of Rumen Microbe–Muscle–Liver Interactions
by Haibo Wang, Jinshun Zhan, Shengguo Zhao, Haoyun Jiang, Haobin Jia, Yue Pan, Xiaojun Zhong and Junhong Huo
Microorganisms 2025, 13(4), 943; https://doi.org/10.3390/microorganisms13040943 - 19 Apr 2025
Viewed by 917
Abstract
Through an integrated multi-omics analysis of rumen microbial communities, muscle transcriptomes, metabolic profiles, and liver metabolic profiles, this study systematically compared high- and low-performing lambs to elucidate their divergent effects on meat quality attributes and growth development. A total of 100 male lambs [...] Read more.
Through an integrated multi-omics analysis of rumen microbial communities, muscle transcriptomes, metabolic profiles, and liver metabolic profiles, this study systematically compared high- and low-performing lambs to elucidate their divergent effects on meat quality attributes and growth development. A total of 100 male lambs with similar birth weight (3.07 ± 0.06 kg) were selected within 72 h. All test lambs were synchronized weaning at 45 days of age and uniformly fed the same diet (total mixed ration) in the same pen until 180 days of age, with ad libitum access to food and water throughout this period. Subsequently, the eight lambs with the highest (HADG) and lowest (LADG) average daily gains were slaughtered for performance evaluation and multi-omics analysis. This study found that HADG lambs increased body weight, muscle fiber diameter, eye muscle area, improved amino acid (histidine, arginine, valine, isoleucine, essential amino acid/total amino acid, and essential amino acid/nonessential amino acid), and fatty acid (linoleic acid, behenic acid, and arachidonic acid) composition enhanced rumen enzymes (pepsase, lipase, xylanase, amylase, and carboxymethyl cellulose) and promoted efficient fermentation (p < 0.05). Analysis of microbial populations indicated a notable increase in Prevotella levels within the rumen of HADG lambs. Furthermore, the rumen markers Schwartzia and Streptococcus exhibited significant correlations with differential meat quality traits. Analysis of the muscle transcriptome indicated a significant correlation between the turquoise module and host phenotypes, particularly body weight. Additionally, muscle metabolism is primarily concentrated within the black module; however, it exhibits a significant correlation with the host body phenotype in the yellow module (p < 0.05). Moreover, liver metabolites, rumen microbes, host phenotype, and muscle transcripts were significantly correlated (p < 0.05). In conclusion, the interactions among rumen microbes, muscle, and liver in lambs promote rumen fermentation, which in turn regulate muscle transcriptional activity and modify metabolic profiles in both the liver and muscle. Moreover, PCK1, SPP1, FGF7, NR4A1, DUSP5, GADD45B, etc., can be candidate genes for muscle growth and development. This finding provides a theoretical basis for further exploiting the production potential of Hu lambs. Full article
(This article belongs to the Section Veterinary Microbiology)
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20 pages, 3664 KB  
Review
‘Geophagy’ and Clay Minerals: Influencing Ruminal Microbial Fermentation for Methane Mitigation
by Zubaer Hosen, Md. Rashidul Islam, Ravi Naidu and Bhabananda Biswas
Microorganisms 2025, 13(4), 866; https://doi.org/10.3390/microorganisms13040866 - 10 Apr 2025
Cited by 2 | Viewed by 2458
Abstract
Methane is a greenhouse gas with high warming potential, and ruminants like cattle and sheep are a major source of its emission. In the rumen, the first stomach compartment, diverse microorganisms and fauna live, including archaea, bacteria, protozoa, nematodes, and fungi. They participate [...] Read more.
Methane is a greenhouse gas with high warming potential, and ruminants like cattle and sheep are a major source of its emission. In the rumen, the first stomach compartment, diverse microorganisms and fauna live, including archaea, bacteria, protozoa, nematodes, and fungi. They participate in complex fermentation processes. During rumen fermentation, various gases are produced, dominantly hydrogen and carbon dioxide. In methanogenesis, methanogens utilize these two gases to produce methane as a byproduct, which burps out into the atmosphere. Therefore, interfering with this methanogenesis is a promising way of reducing methane. Supplementing feed containing clay minerals could be one of method to do so as ruminants naturally consume them as they graze, often called “geophagy”. This review discusses the role of clay minerals in enteric methane abatement, emphasizing the clay–microbial interaction in the rumen. In these interactions, clay minerals also serve as a carrier for other chemicals and influence microbial attachment. Elemental dissolution and cations from clay mineral and their buffering capacity can further influence microbial dynamics in rumen fluids. By combining insights from microbiology, soil science, and animal nutrition, this review provides an interdisciplinary view of rumen interactions. Findings from this review can help to develop a low-cost and safe clay feed supplement to reduce livestock methane emissions. Full article
(This article belongs to the Special Issue Microbiome Research for Animal, Plant and Environmental Health)
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16 pages, 1607 KB  
Article
Effects of Rice Straw Variety on Rumen Fermentation, Bacterial Community, and Metabolite Profile
by Tanghui Long, Yashi Li, Mengying Li, Kehui Ouyang, Mingren Qu, Ke Pan and Qinghua Qiu
Agriculture 2025, 15(7), 739; https://doi.org/10.3390/agriculture15070739 - 30 Mar 2025
Viewed by 893
Abstract
The objective of this study was to investigate the effects of different rice varieties on in vitro rumen fermentation characteristics, microbial community, and metabolite profile of rice straw. The inbred variety Zhongjiazao 17 (Z17) and the hybrid variety Ruiliangyou 1053 (R1053) were selected [...] Read more.
The objective of this study was to investigate the effects of different rice varieties on in vitro rumen fermentation characteristics, microbial community, and metabolite profile of rice straw. The inbred variety Zhongjiazao 17 (Z17) and the hybrid variety Ruiliangyou 1053 (R1053) were selected as the two test varieties. Each variety was subjected to in vitro rumen fermentation for 72 h, with five replicates conducted for each. The results showed that R1053 had higher values in total gas production, in vitro dry matter digestibility, total volatile fatty acids (VFA), and individual VFA compared to Z17. Additionally, R1053 exhibited higher values in rumen microbial diversity indices, including Chao1, observed species, PD whole tree, and Shannon index, compared to Z17. The Z17 group had higher relative abundances of Verrucomicrobiota, Chloroflexi, Saccharofermentans, Probable genus 10, and Lachnospiraceae AC2044 group. In contrast, the R1053 group had higher relative abundance of Patescibacteria. Analysis of similarities (ANOSIM) confirmed a significant difference between the Z17 and R1053 groups (R = 0.8160, p = 0.005). Metabolomic analysis identified a total of nine differential metabolites, with four upregulated in the Z17 group and five upregulated in the R1053 group. This study demonstrates that the hybrid variety R1053 has a higher utilization value than the inbred variety Z17, which is closely associated with rumen microbes and metabolites. This study provides new insights into the efficient utilization of rice straw in ruminants from microbiological and metabolomic perspectives. Full article
(This article belongs to the Section Farm Animal Production)
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26 pages, 2747 KB  
Article
Physiological and Microbial Community Dynamics in Does During Mid-Gestation to Lactation and Their Impact on the Growth, Immune Function, and Microbiome Transmission of Offspring Kids
by Haidong Du, Kenan Li, Wenliang Guo, Meila Na, Jing Zhang, Jing Zhang and Renhua Na
Animals 2025, 15(7), 954; https://doi.org/10.3390/ani15070954 - 26 Mar 2025
Viewed by 567
Abstract
This study investigated changes in physiological processes and rumen microbial communities in does from mid-gestation to lactation and identified potential associations between these physiological changes and the rumen microbiome. Additionally, we studied the transmission mechanisms of microorganisms between the dam and offspring. Our [...] Read more.
This study investigated changes in physiological processes and rumen microbial communities in does from mid-gestation to lactation and identified potential associations between these physiological changes and the rumen microbiome. Additionally, we studied the transmission mechanisms of microorganisms between the dam and offspring. Our study demonstrates significant changes in maternal physiological metabolism, immune status, and rumen microbiota from mid-pregnancy through lactation. We identified potential associations between these physiological changes and the rumen microbiome. Moreover, the findings highlight that alterations in maternal physiological metabolism and immune status significantly influence the growth and immune development of offspring kids. Additionally, we observed that the maternal microbiota serves as a key source of gastrointestinal microbial communities in young animals, with early colonization of maternally derived microbes in the offspring’s gastrointestinal tract playing a role in shaping their immune system development. The results for primary outcomes are as follows: The serum levels of estrogen and progesterone in pregnant does were greater than those observed during lactation, while the concentration of growth hormone, triiodothyronine, and glucose exhibited an upward trend during lactation. During late gestation, the serum IL-10 concentration in does decreased, while the TNF-α concentration increased. Additionally, on day 140 of gestation, does showed a significant decrease in IgG, total protein, and globulin levels. From mid-gestation to lactation, the abundance of dominant phyla and genera, including Firmicutes, Bacteroidetes, Patescibacteria, Bacteroidales_RF16_group, Clostridia_UCG-014, RF39, and Eubacterium_ventriosum_group, in the rumen of does underwent significant changes. LEfSe analysis identified a series of marker microorganisms in the rumen of does at different physiological stages. A correlation was observed between these dominant bacteria and the serum physiological indicators of the does. Notably, rumen volatile fatty acids also exhibited a correlation with serum physiological indicators. In addition, serum physiological indicators of does were significantly correlated with the growth and immune indicators of their kids. Microbiological origin analysis revealed that the gastrointestinal microbiome of kids primarily originated from the rumen, birth canal, and milk of does. Further analysis identified a correlation between the kids’ serum immunometric indicators and certain gastrointestinal microorganisms. In particular, the jejunum microbiota of 28-day-old lactating kids, including Alysiella, Neisseria, and Muribaculaceae, showed a significant positive correlation with serum IL-6 and IL-10 levels. Meanwhile, these genera were dominant in the saliva and milk of does, suggesting a direct microbial transfer from dam to offspring. These microbial communities may play a significant role in modulating the metabolism and immune responses of the offspring, thereby influencing their immune system development. Full article
(This article belongs to the Section Small Ruminants)
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13 pages, 2738 KB  
Article
Metabolomics Study Revealed the Effects of CaO-Treated Maize Straw on the Rumen Metabolites
by Hui Wang, Mingjun Shi, Zhanxia Ma, Xuewei Zhang, Huiyong Shan, Xiaofeng Xu, Suyu Quan, Junqin Zhang and Yujia Tian
Animals 2025, 15(5), 674; https://doi.org/10.3390/ani15050674 - 26 Feb 2025
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Abstract
As an important limiting factor, lignin hinders the utilization rate of maize straw in ruminants. CaO treatment increases the feed digestibility of maize straw by disrupting the ester bonds between hemicellulose, cellulose, and lignin in maize straw. Our previous research found that CaO [...] Read more.
As an important limiting factor, lignin hinders the utilization rate of maize straw in ruminants. CaO treatment increases the feed digestibility of maize straw by disrupting the ester bonds between hemicellulose, cellulose, and lignin in maize straw. Our previous research found that CaO treatment of corn straw may increase its feed digestibility by altering the rumen microbes’ abundance. This study further investigated the molecular mechanism of CaO treatment to enhance feed utilization and also examined its ongoing effects on rumen metabolites. Rumen fluid was collected to analyze microbial metabolites using liquid chromatography–mass spectrometry (LC–MS) non-targeted metabolomics. Maize straw (moisture content of 60%) treated with four levels of CaO (0%, 3%, 5%, and 7%) was used as the fermentation substrate for a 6 h in vitro culture. Based on the effect of CaO-treated maize straw on the rumen microbial diversity, no significant differences were observed in microbial composition between the 0% and 3% treatment groups or between the 5% and 7% treatment groups. However, the microbial structure of the 0% and 3% treatment groups differed from that of the 5% and 7% groups. Therefore, the four levels were divided into a low-efficiency group (LE group: 0% and 3% levels) and a high-efficiency group (HE group: 5% and 7% levels) for principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA). Compared with the LE group, most of the ruminal metabolites that showed increased levels in the HE group were products of lignin degradation. Among these differential metabolites, Dihydro-3-coumaric acid had a significant positive correlation with Prevotella and fermentation indicators like acetate, propionate, and butyrate. KEGG analysis showed differential metabolites were primarily enriched in the amino acid metabolism, tryptophan metabolism, phenylalanine, tyrosine, and tryptophan biosynthesis, and cyanoamino acid metabolism. The higher CaO concentration in the HE group effectively disrupted most covalent bonds with lignin, significantly enhancing cellulose degradation and ultimately supporting improved rumen metabolism. Full article
(This article belongs to the Section Animal Physiology)
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20 pages, 3628 KB  
Article
In Vitro Investigation of the Effects of Bacillus subtilis-810B and Bacillus licheniformis-809A on the Rumen Fermentation and Microbiota
by Raphaële Gresse, Bruno Ieda Cappellozza, Didier Macheboeuf, Angélique Torrent, Jeanne Danon, Lena Capern, Dorthe Sandvang, Vincent Niderkorn, Giuseppe Copani and Evelyne Forano
Animals 2025, 15(4), 476; https://doi.org/10.3390/ani15040476 - 7 Feb 2025
Cited by 3 | Viewed by 2585
Abstract
Direct-fed microbials (DFMs) have shown the potential to improve livestock performance and overall health. Extensive research has been conducted to identify new DFMs and understand their mechanisms of action in the gut. Bacillus species are multifunctional spore-forming bacteria that exhibit resilience to harsh [...] Read more.
Direct-fed microbials (DFMs) have shown the potential to improve livestock performance and overall health. Extensive research has been conducted to identify new DFMs and understand their mechanisms of action in the gut. Bacillus species are multifunctional spore-forming bacteria that exhibit resilience to harsh conditions, making them ideal candidates for applications in the feed industry and livestock production. This study investigates the mode of action of B. licheniformis and B. subtilis in the rumen using diverse in vitro techniques. Our results revealed that both strains germinated and grew in sterile rumen and intestinal contents from dairy cows and bulls. Gas composition analysis of in vitro cultures in a medium containing 40% rumen fluid demonstrated that germination of B. licheniformis and B. subtilis strains reduced oxygen levels, promoting an anaerobic environment favorable to rumen microbes. Enzymatic activity assays showed that B. licheniformis released sugars from complex substrates and purified polysaccharides in filtered rumen content. Additionally, the combination of B. licheniformis and B. subtilis survived and grew in the presence of a commercial monensin dose in rumen fluid media. The effects of B. licheniformis and B. subtilis on rumen fermentation activity and microbiota were studied using an in vitro batch fermentation assay. In fermenters that received a combination of B. licheniformis and B. subtilis, less CO2 was produced while dry matter degradation and CH4 production was comparable to the control condition, indicating better efficiency of dry matter utilization by the microbiota. The investigation of microbiota composition between supplemented and control fermenters showed no significant effect on alpha and beta diversity. However, the differential analysis highlighted changes in several taxa between the two conditions. Altogether, our data suggests that the administration of these strains of Bacillus could have a beneficial impact on rumen function, and consequently, on health and performance of ruminants. Full article
(This article belongs to the Section Cattle)
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26 pages, 5013 KB  
Article
Anti-Methanogenic Potential of Seaweeds and Impact on Feed Fermentation and Rumen Microbiome In Vitro
by Pradeep Kumar Malik, Atul Purshottam Kolte, Shraddha Trivedi, Govindan Tamilmani, Archit Mohapatra, Shalini Vaswani, Johnson Belevendran, Artabandhu Sahoo, Achamveetil Gopalakrishnan and Raghavendra Bhatta
Microorganisms 2025, 13(1), 123; https://doi.org/10.3390/microorganisms13010123 - 9 Jan 2025
Cited by 1 | Viewed by 1560
Abstract
A series of in vitro studies were conducted to explore the anti-methanogenic potential of five seaweeds collected from the Indian sea and to optimize the level(s) of incorporation of the most promising seaweed(s) into a straw and concentrate diet to achieve a significant [...] Read more.
A series of in vitro studies were conducted to explore the anti-methanogenic potential of five seaweeds collected from the Indian sea and to optimize the level(s) of incorporation of the most promising seaweed(s) into a straw and concentrate diet to achieve a significant reduction in methane (CH4) production without disturbing rumen fermentation characteristics. A chemical composition analysis revealed a notable ash content varying between 55 and 70% in seaweeds. The crude protein content was highly variable and ranged between 3.25 and 15.3% of dry matter. Seaweeds contained appreciable concentrations of tannins and saponins. Among the seaweeds, Spyridia filamentosa exhibited significantly higher CH4 production, whereas the percentage of CH4 in total gas was significantly lower in the cases of Kappaphycus alvarezii and Sargassum wightii. The ranking of seaweeds in terms of CH4 production (mL/g OM) is as follows: Sargassum wightii < Kappaphycus alvarezii < Acanthophora specifera < Padina gymnospora < Spyridia filamentosa. A remarkable decrease of 31–42% in CH4 production was recorded with the incremental inclusion of Kappaphycus alvarezii at levels of 3–5% of the dry matter in the diet. The addition of Sargassum wightii led to a significant decrease of 36–48% in CH4 emissions when incorporated at levels of 4–5% of dry matter, respectively. The findings of this study revealed a significant decrease in the numbers of total protozoa and Entodinomorphs, coupled with increasing abundances of sulfate-reducing microbes and minor methanogens. Metagenome data revealed that irrespective of the seaweed and treatment, the predominant microbial phyla included Bacteroidota, Bacillota, Pseudomonadota, Actinomycetota, Fibrobacterota, and Euryarchaeota. The prevalence of Methanobrevibacter was similar across treatments, constituting the majority (~79%) of the archaeal community. The results also demonstrated that the supplementation of Kappaphycus alvarezii and Sargassum wightii did not alter the feed fermentation pattern, and therefore, the reduction in CH4 production in the present study could not be attributed to it. Animal studies are warranted to validate the extent of reduction in CH4 production and the key processes involved by supplementation with Kappaphycus alvarezii and Sargassum wightii at the recommended levels. Full article
(This article belongs to the Special Issue Dietary and Animal Gut Microbiota)
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