Wheat straw (WS) is an abundant crop residue with considerable potential as a ruminant feed; however, its utilization is severely constrained by a recalcitrant lignocellulosic structure. This study evaluated the effects of five alkali-resistant cellulolytic bacterial inoculants on the structural carbohydrate composition and in vitro ruminal fermentation characteristics of ammoniated wheat straw. A 6 × 3 factorial arrangement was employed, with six treatments (ammoniated WS as a control and five cellulolytic bacterial strains: X67, C72, S87, D100, and X107) and three storage durations (7, 14, and 21 days). The results showed that the bacterial treatments caused moderate losses of dry matter (DM), neutral detergent fiber (NDF), acid detergent fiber (ADF), hemicellulose, and cellulose at 7 and 14 days. The X107 treatment exhibited the lowest hemicellulose content, 2.1% lower than the ammoniated control. After 48 h of in vitro incubation, all bacterial treatments significantly increased potential gas production, 48 h methane production, in vitro DM digestibility (IVDMD), and in vitro NDF digestibility (IVNDFD). The S87 treatment achieved the highest IVDMD and IVNDFD, exceeding the ammoniated control by 14.9% and 32.5%, respectively, at 7 days (p < 0.05). All bacterial treatments maintained relatively high total volatile fatty acid (VFA) concentrations. Furthermore, the bacterial treatments increased the relative proportions of ruminal cellulolytic microbes at 7 and 14 days. By 21 days, no significant differences were observed in DM loss or microbial proportions among treatments. These findings indicate that the application of cellulolytic bacterial additives, with appropriate selection of bacterial strain and storage duration, exerts synergistic positive effects on the feeding value of alkali-pretreated wheat straw. The S87 treatment with a 7-day storage duration proved most effective.

Inefficient enzyme-microbe synergy hinders lignocellulosic waste valorization. This study developed an integrated system employing a machine learning-driven workflow, combining CatPred for kinetic prediction and ESMFold-AlphaFold2 for 3D structure optimization, to engineer a xylanase variant with superior binding affinity (-9.30 kcal/mol). The recombinant enzyme achieved an 8.67-fold increase in catalytic efficiency, elevating xylose release from 0.35 to 3.37 g/L. Coupled with Pediococcus pentosaceus, a dual-pathway conversion system for alfalfa waste was established. While enzymatic treatment alone yielded 1.43 g/kg DM xylose, the synergistic system (PE) enhanced lactic acid production by 55.7% (to 54.96 g/kg DM), reduced hemicellulose by 68.68%, and increased crude protein by 25.42%. PE fermentation enriched P. pentosaceus dominance (62.07%) and upregulated key genes in xylose metabolism and lactate synthesis. Techno-economic analysis confirmed a 2.3-fold increase in economic returns, establishing a viable paradigm for sustainable lignocellulose biorefining.

The limited bioavailability of essential amino acids (EAAs) in alfalfa silage, caused by proteolysis during ensiling and extensive ruminal degradation, restricts its protein nutritional value for ruminants. This study evaluated the individual and combined effects of lactic acid bacteria (LAB) and grape seed tannins (G) on the dynamic fate of essential amino acids (EAAs) throughout ensiling, ruminal fermentation, and intestinal digestion. LAB treatment accelerated acidification and improved EAA preservation during ensiling but, importantly, increased ruminal degradation of most EAAs. In contrast, tannins alone reduced ruminal degradation of key EAAs. Compared with the control, the combined LG treatment enhanced the retention of EAAs and reduced NH3-N concentration by 25.5

Leymus chinensis (sheepgrass), a dominant perennial grass of the Eurasian Steppe, is a crucial source of carbohydrates and energy for ruminants. However, the lignocellulose recalcitrance severely limits its digestibility. Here, we targeted xylan, a major hemicellulose interacting with cellulose and lignin in cell wall. To improve digestibility, we knocked out two IRREGULAR XYLEM 14-LIKE (LcIRX14L) genes, which encode key enzymes for xylan biosynthesis. The Lcirx14L knockout mutants exhibited reduced xylan molecular weight but produced similar amounts of biomass to wild-type plants. Simon's staining and cellulase binding assays indicated enhanced cellulose accessibility in the Lcirx14L mutant. In addition, the Lcirx14L plant showed increased saccharification efficiency, dry matter and NDF digestibility, and production of volatile fatty acids by in vitro fermentation. This study thus provides novel insight for engineering forage grasses with both high yield and quality by modifying xylan polymerization.

Fermentation technology has long been applied in animal nutrition worldwide, with its application primarily focused on animal feed [...]

Astragalus polysaccharide possesses promising potential as a natural non-antibiotic growth promoter for ruminant production. This study investigated the effects of dietary supplementation with coated Astragalus polysaccharide (CAPS) on growth performance, nutrient digestibility, rumen fermentation, serum metabolites, and antioxidant capacity in Angus beef cattle. A total of 60 late-fattening Angus bulls (Body weight: 650 +/- 29.6 kg) were randomly divided into four treatment groups. Bulls in the four treatment groups were fed a basal diet supplemented with 0, 0.9, 1.8, and 2.7 g of CAPS (Control, LCAPS, MCAPS, and HCAPS, respectively) per kg of dietary dry matter (DM) for 60 days. Increasing dietary CAPS supplementation linearly increased average daily gain (ADG) and nutrient digestibility while quadratically decreasing feed-to-gain ratio (F/ G), with MCAPS treatment resulting in the highest ADG and nutrient digestibility and the lowest F/G. MCAPS and HCAPS treatments stimulated ruminal total short-chain fatty acid production and shifted the fermentation pattern toward propionate formation. Feeding CAPS linearly increased the abundance of Rumincoccus albus, Rumincoccus flavefaciens, Ruminobacer amylophilus, and Prevotella ruminicola and the activity of carboxymethyl-cellulase, laccase, alpha-amylase, and protease. Blood urea nitrogen and serum cholesterol concentrations were decreased linearly with CAPS supplementation. Moreover, the serum oxidative stress parameters showed that 1.8 and 2.7 g/kg DM of CAPS addition increased total superoxide dismutase, glutathione peroxidase, and catalase activities and decreased malondialdehyde contents. These findings demonstrate that supplementing the diet with 1.8 g/kg DM of CAPS optimally enhanced growth performance, rumen function, metabolic efficiency, and serum antioxidant status in Angus bulls.

This study proposes an ensiling-biorefinery-bioethanol process for valorizing corn stover (CS) into value-added lactic acid (LA) and bioethanol. The effects of ensiling performance modulation by employing bio-additives on the optimization of coupling biorefinery and bioethanol production were investigated. The CS was either ensiled without additives (CON) or with lignocellulosic enzymes derived from liquid fermentation by Pleurotus ostreatus (LEY), a lactic acid bacteria inoculant containing Lactobacillus plantarum and Pediococcus pentosaceus (LPP), and a combination of LEY and LPP (LEL). In the ensiling process, the addition of additives promoted homolactic fermentation by enriching Lactiplantibacillus and lignocellulose degradation, elevating LA production and reducing organic dry matter losses in CS silage. The synergistic modulation of lignocellulosic enzymes and lactic acid bacteria (LAB) enhanced the silage-based biorefinery efficiency, as evidenced by a 145 % increase in LA concentration in the liquid phase. The remaining silage solids achieved 99% cellulose recovery, and 44.7 and 46.8% increase in the glucan and xylan conversion, respectively. Simultaneous saccharification and cofermentation of LEL-fortified silage solids at 20 % loading exhibited the highest ethanol titer of 40.9 g/L with a yield of 70.2%. Besides, mass balance revealed that 1 kg fresh CS can generate 15.9 g LA and 59.2 g ethanol based on LEL-fortified ensiling-biorefinery-bioethanol process. These findings demonstrate the feasibility of LELfortified ensiling-biorefinery-bioethanol approach for the sustainable conversion of CS into LA and bioethanol.

Abstract Electrocatalytic nitrite reduction for ammonia synthesis offers a novel green pathway to overcome limitations of the Haber–Bosch process, though it remains constrained by issues such as sluggish adsorption/desorption kinetics of reaction intermediates. This study employs waste peanut shells as feedstock to synthesize FeP2/PS nanocatalysts in situ via single-step high-temperature calcination. This catalyst enhances the active surface area through the incorporation of a biochar support and phosphorus (P) elements. Simultaneously, the introduction of phosphorus atoms and their electronegativity induces a directed electron transfer from iron to phosphorus within FeP2. The resulting FeP2/PS exhibits a Faradaic efficiency of 98.94% in the nitrite reduction reaction (NO2–RR), with an ammonia yield of 308.459 μmol h–1 cm–2. Performance remained stable after 15 cycles. Based on density functional theory (DFT) calculations, the P sites in FeP2/PS act as bridges for electron and proton transport, effectively mediating electron transfer and charge supply to the Fe active sites, and optimizing the electronic structure of the Fe sites, thereby greatly enhancing the catalyst’s adsorption of nitrite (NO2–) and key reaction intermediates, lowering the reaction energy barrier, and accelerating the NO2– reduction kinetics, which significantly improves the yield of electrocatalytic ammonia synthesis.

This study evaluated the influences of coated folic acid (CFA) and folic acid (FA) on lactation performance, apparent digestibility, rumen volatile fatty acid (VFA) production, blood metabolism, and hepatic lipid content in cows. A total of 140 Holstein cows were allocated to seven groups in a randomized block design. Cows in the control received no addition, those in the in low CFA (LCFA), medium CFA (MCFA), and high CFA (HCFA) groups received CFA at 135, 270, and 405 mg FA/d, and those in the low FA (LFA), medium FA (MFA), and high FA (HFA) groups received FA at 135, 270, and 405 mg/d. The experiment began 5 weeks before calving to 6 weeks after calving. When increasing the level of CFA, the fat-corrected milk (FCM), fat and protein yields, de novo fatty acid content, and feed efficiency increased linearly. A linear increase was observed for nutrient digestibility and ruminal total VFAs. The blood total protein, albumin, superoxide dismutase, glutathione peroxidase, and folate increased linearly, but blood non-esterified fatty acids and β-hydroxybutyric acid and hepatic lipids decreased linearly. When increasing the level of FA, the FCM and milk fat yields increased linearly, but the rumen total VFA increased quadratically. Compared with MFA, cows receiving MCFA had a greater milk yield and lower hepatic lipids. Overall, the addition of CFA increased the milk yield and decreased the hepatic lipid content in cows.

This study evaluated the effects of rumen-protected chromium-nicotinic acid (RPCNA) supplementation on lactation performance, nutrient digestion, ruminal fermentation, serum biochemical parameters and antioxidant in lactating water buffaloes under conditions of a critical thermal comfort index (TCI). Healthy lactating water buffaloes (milk yield = 5.96 ± 0.21 kg; parity = 2.96 ± 0.15, mean ± SD) were randomly assigned to four groups, which were the control (without RPCNA), and three treatment groups designated as RPCNA2, RPCNA4, and RPCNA6, with 0, 2, 4, and 6 mg/(d·head) of RPCNA, respectively. During a 56-day experimental period, the average temperature was 29 °C with an average TCI of 40.60, indicating a critical state of environmental stress. The results showed that the addition of RPCNA to the diet had no significant effect on the apparent digestibility of nutrients, lactation performance, and dry matter intake in lactating water buffaloes. However, RPCNA supplementation positively influenced rumen fermentation, reducing ammonia nitrogen (NH3-N) concentrations and promoting microbial protein synthesis. Supplementation with RPCNA4 and RPCNA6 decreased rumen NH3-N levels by 48.3% and 36.4%, respectively, while RPCNA4 increased isobutyrate concentrations. This demonstrates that the coating technology does not provide 100% rumen protection. Serum analysis revealed that RPCNA significantly increases total antioxidant capacity (T-AOC). Additionally, the supplementation of 4 mg/(d·head) of RPCNA, improved serum glutathione peroxidase (GSH-Px) activity and reduced malondialdehyde (MDA) levels. These findings suggest that moderate doses of RPCNA supplementation can improve antioxidant status and rumen nitrogen metabolism in lactating water buffaloes under critical state, without significantly altering milk production or composition.

To enhance hemicellulose utilization in alfalfa silage, Lactococcus lactis (NE) and Pediococcus pentosaceus (PE) expressing xylanase were engineered. The effects of recombinant LAB on hemicellulose utilization, fermentation characteristics, and microbiota diversity during alfalfa ensiling were investigated. The recombinant strains exhibited a 2.35-fold higher xylan degradation efficiency in comparison to the wild-type strains. Recombinant PE treatment significantly improved silage quality, with lactic acid (LA) production surging 149%, pH declining from 5.9 to 4.7 during the early stage (3 days), and hemicellulose degradation increased by 39.5% after 60 days of ensiling. Correlation analysis and KEGG pathway analysis confirmed that the relative abundance of Pediococcus affects the changes in the LA, hemicellulose, and carbohydrate catabolism. Compared to the control group, recombinant PE treatment increased in vitro dry matter digestibility (IVDMD, 20.2%), neutral detergent fiber digestibility (IVNDFD, 12.1%), and acid detergent fiber digestibility (IVADFD, 18.0%). Thus, the engineered strain secretes xylanase to enhance silage fermentation and fiber digestibility.

This study examined the effects of alfalfa silage versus alfalfa hay in a total mixed ration (TMR) on milk yield, rumen fermentation, and nutrient digestibility in dairy cows. Hydrolyzed tannins (HT) were supplemented individually to assess changes. Thirty-two multiparous Holstein cows (DIM: 94 ± 8 d; milk yield: 41 ± 2 kg) were assigned to four treatments in a 2 × 2 factorial design: basal diet (alfalfa hay, H, or alfalfa silage, S) and additive (control, C, or 100 g/d HT, T). Production performance, rumen fermentation, nutrient digestibility, and blood metabolites were evaluated. Compared with group H, group S had a 0.16% higher milk protein percentage and significantly higher fat-corrected milk yield, milk fat percentage, fat-to-protein ratio, total solids, and milk urea nitrogen. After feeding, the ST group had increased ruminal pH. HT supplementation significantly decreased ruminal NH3-N levels (p < 0.05) and increased microbial crude protein (MCP) content (p < 0.05). Group H showed no significant changes, and the effects of HT were less evident in hay-fed cows than in silage-fed cows. In summary, alfalfa silage feeding increased ruminal microbial populations, while HT supplementation mitigated the post-feeding decline in ruminal pH. Considering the relatively small sample size (n = 32), the results should be viewed as indicative rather than conclusive, and future studies with larger cohorts will be valuable to confirm and extend these findings.

To develop sustainable strategies for mitigating ruminal methanogenesis and improving nitrogen efficiency in dairy systems, this study investigated how low-dose tannic acid (T), tea polyphenols (TP), and their combination (T+TP; 50:50) modulate rumen microbiota and function. A sample of Holstein cows were given four dietary treatments: (1) control (basal diet); (2) T (basal diet + 0.4% DM tannic acid); (3) TP (basal diet + 0.4% DM tea polyphenols); and (4) T+TP (basal diet + 0.2% DM tannic acid + 0.2% DM tea polyphenols). We comprehensively analyzed rumen fermentation, methane production, nutrient digestibility, milk parameters, and microbiota dynamics. Compared with the control group, all diets supplemented with additives significantly reduced enteric methane production (13.68% for T, 11.40% for TP, and 10.89% for T+TP) and significantly increased milk protein yield. The crude protein digestibility significantly increased in the T group versus control. The results did not impair rumen health or fiber digestion. Critically, microbiota analysis revealed treatment-specific modulation: the T group showed decreased Ruminococcus flavefaciens abundance, while all tannin treatments reduced abundances of Ruminococcus albus and total methanogens. These microbial shifts corresponded with functional outcomes—most notably, the T+TP synergy drove the largest reductions in rumen ammonia-N (34.5%) and milk urea nitrogen (21.1%). Supplementation at 0.4% DM, particularly the T+TP combination, effectively enhances nitrogen efficiency and milk protein synthesis while reducing methane emissions through targeted modulation of key rumen microbiota populations, suggesting potential sustainability benefits linked to altered rumen fermentation.

Coated sodium selenite (CSS) is a rumen-protected selenium supplement that can improve selenium status and lactation performance in buffalo. This study investigated the effects of CSS supplementation on milk yield, rumen fermentation, digestibility, blood biochemical parameters, and antioxidant capacity in 28 dairy hybrid buffaloes (Murrah × local breed; milk yield = 5.96 ± 0.21 kg/d; parity = 2.96 ± 0.15, mean ± SD). The buffaloes were randomly allocated into four groups: control (basal diet), low CSS (LCSS, basal diet + 0.1 mg/kg CSS), medium CSS (MCSS, basal diet + 0.15 mg/kg CSS), and high CSS (HCSS, basal diet + 0.2 mg/kg CSS). The trial included a 7-day adaptation period followed by a 60-day experimental period. Compared with the control group, the LCSS group showed significant increases in rumen acetic acid, propionic acid, and total volatile fatty acid contents; milk yield, milk fat percentage, and 4% standard milk yield; neutral detergent fiber digestibility; and antioxidant capacity. These results demonstrate that supplementing 0.1 mg/kg CSS improves rumen fermentation efficiency and cellulose digestibility, thereby enhancing the antioxidant capacity and lactation performance of dairy buffaloes.

Aflatoxin B1 (AFB1) and zearalenone (ZEN) are two common mycotoxins found in grain feed, which cause substantial economic losses. This study employed molecular docking predictions, codon optimization, signal peptide modification, and a nisin-controlled gene expression system. The heterologous expression of acid ligninase (LigX) in food-grade Lactococcus lactis was achieved for the first time to degrade these toxins. Molecular docking experiments verified the high-affinity interaction between LigX and mycotoxins. Key residues within the enzyme engage the toxins via cation-π contacts, π-π stacking, and an extensive hydrogen-bond network. The lowest computed binding free energies are -7.5 kcal mol-1 for AFB1 and - 7.1 kcal mol-1 for ZEN, indicating highly favorable complex formation. Codon optimization and signal peptide engineering successfully increased the extracellular LigX expression efficiency by 34 % in Lactococcus lactis to a high level (up to 78 U/L). At 1 ng/mL nisin, the extracellular enzyme production of the nisin control system increased by 480 %. The SGM17 medium contained Mg2+ and Ca2+ (as coenzyme activator), which effectively increased the degradation rates of AFB1 (14.7 %) and ZEN (73.9 %) under 48 h. Tests revealed the optimized recombinant strain degraded 41 % of AFB1 and 97 % of ZEN under low pH 6 conditions. LC-MS analysis predicted key degradation products and pathways: AFB1 underwent furan ring epoxidation and lactone cleavage, while ZEN was metabolized via hydroxylation, lactone hydrolysis, and oxidative polymerization-pathways that disrupt toxic structures and reduce toxicity. This study demonstrates the potential of engineered L. lactis system as a promising biocontrol strategy for mycotoxin mitigation in feed and food.

Aflatoxin B1 (AFB1) poses a serious threat to the health of cattle and is strictly monitored in their diets. Rumen microorganisms in cattle have the ability to degrade AFB1, but this capability is limited. Enhancing the degradation capacity of the original rumen microecology for AFB1 is a novel approach. From a molecular perspective,indoleacetic-3-acid (IAA) promotes the expression of cytochromes, which can improve the degradation of AFB1. Therefore, this study aims to investigate the impact of different concentrations of IAA on the degradation of AFB1 by the rumen microecology. Experiments used rumen fluid from three adult cows as donors, and the cows were all fed the same total mixed ration. Rumen fluid was collected from these three cannulated cows before morning feeding to prepare in vitro fermentation fluid. The experiments used a completely randomized design, with each treatment repeated four times. The results showed that as the fermentation time increased, the content of AFB1 gradually decreased, with a degradation rate of up to 75.73% after 24 h. AFB1 altered the rumen fermentation pattern, with a significant reduction in the content of acetic acid (P<0.05) and a significant decrease in the acetic acid to propionic acid ratio (P<0.05). It also affected the rumen microecology, causing a significant reduction in the abundance of Ruminococcus amylophilus, Prevotella ruminicola, and Fusobacterium succinogenes (P<0.05). In addition, this study found that with the increase in the amount of IAA added, the content of AFB1 in the rumen gradually decreased. IAA enhances the degradation capacity of the original rumen microecology for AFB1, and the addition of IAA alleviates the impact of AFB1 on Ruminococcus amylophilus, Prevotella ruminicola, and Fusobacterium succinogenes in the rumen (P<0.05). Moreover, the addition of IAA can promote the stability of the rumen microecology, with significantly higher acetic acid and acetic acid to propionic acid ratios in the fermentation fluid compared to the non-added group (P<0.05). In summary, the addition of IAA can improve the degradation capacity of the rumen microecology for AFB1, providing a new solution for alleviating the impact of AFB1 on animal health. ### Competing Interest Statement The authors have declared no competing interest.

Aflatoxin B1 (AFB1) has been recognized as a serious health risk for ruminant animals. From a molecular perspective, indole-3-acid (IAA) possesses the potential to enhance the removal of AFB1 by rumen microbiota. Therefore, this study aims to investigate the impact of different concentrations of IAA on the removal of AFB1 by rumen microbiota using an in vitro technique. Experiment 1: interaction between AFB1 and rumen fermentation. Experiment 2: The study used a randomized design with five IAA levels (0, 15, 150, 1,500, and 7,500 mg/kg) to examine the effect of IAA on AFB1 removal and its impact on rumen fermentation. The results showed: (1) the content of AFB1 gradually decreased, removal rate of up to 75.73% after 24 h. AFB1 exposure altered the rumen fermentation pattern, with significantly decreased in the acetic acid/propionic acid ratio (p < 0.05). It significantly reduced the relative proportions of R. amylophilus, P. ruminicola, and F. succinogenes (p < 0.05). (2) As the content of IAA increased, AFB1 exposure decreased. A total of 15 and 150 mg/kg IAA significantly mitigated the negative impact of AFB1 on key rumen bacteria (R. amylophilus, P. ruminicola and F. succinogenes), increased acetate levels and acetate/propionate ratio (p < 0.05). However, 1,500 mg/kg IAA lowered levels of propionate and isovalerate, adversely affected enzyme activities (pectinase, xylan and Carboxymethyl-cellulase) and relative proportions of microbiota (R. flavefaciens, P. ruminicola and F. succinogenes). In conclusion, IAA significantly removed AFB1, and in the range of 150 mg/kg of IAA reduced the negative effects of AFB1 on in vitro fermentation characteristics and fermentation end-products.

BACKGROUND Considering the high energy demand of lactation and the potential of guanidinoacetic acid (GAA) addition on the increase in creatine supply for cows, the present study investigated the effects of 0, 0.3, 0.6 and 0.9 g kg(-1) dry matter (DM) of GAA supplementation on lactation performance, nutrient digestion and ruminal fermentation in dairy cows. The study used 40 mid-lactation multiparous Holstein cows and the study duration was 100 days. Results DM intake was not affected, but milk and milk component yields and feed efficiency increased linearly with increasing GAA addition. The total-tract digestibility of DM, organic matter, neutral detergent fibre, acid detergent fibre and non-fibre carbohydrates increased linearly and that of crude protein increased quadratically with increasing GAA addition. When the addition level of GAA increased, ruminal pH, molar percentages of butyrate, isobutyrate and isovalerate and the acetate-to-propionate ratio decreased linearly, and the total volatile fatty acids concentration and propionate molar percentage also increased linearly, whereas the acetate molar percentage and ammonia-N concentration were unaltered. The activities of fibrolytic enzymes, alpha-amylase and protease increased linearly. The populations of total bacteria, fungi, Ruminococcus albus, Fibrobacter succinogenes, Ruminococcus flavefaciens, Ruminobacter amylophilus and Prevotella ruminicola increased linearly, whereas protozoa and methanogens decreased linearly with increasing GAA addition. As for the blood metabolites, concentrations of glucose, urea nitrogen and methionine were unchanged, total protein, albumin, creatine and homocysteine increased linearly, and folate decreased linearly with increasing GAA supply. Conclusion The results of the present study indicate that supplementation of GAA improved milk performance and rumen fermentation in lactating dairy cows. (c) 2022 Society of Chemical Industry.