Huaxin Niu

In this study, three bovine-derived lactic acid bacteria (LAB) strains were isolated from feces of healthy Simmental calves: Lacticaseibacillus paracasei (Lp), Enterococcus hirae (Eh), and Enterococcus faecalis (Ef). Bacterial suspensions were inoculated into pasteurized milk at a 2% inoculation ratio and anaerobically fermented at 37 °C for 24 h to obtain three kinds of LAB-fermented milks: Lacticaseibacillus paracasei fermented milk (LpM), Enterococcus hirae fermented milk (EhM), and Enterococcus faecalis fermented milk (EfM). Their probiotic potential was comprehensively evaluated through a series of in vitro assays, and in vitro colonic anaerobic fermentation was conducted using fecal inoculum from four Simmental calves (three biological replicates per group) to investigate the regulatory effects of these strains on the colonic microbiota and short-chain fatty acids (SCFAs). The three strains exhibited similar growth trends during raw milk fermentation. Compared with Eh and Ef, Lp showed significantly greater utilization of galactooligosaccharides (GOS) and lactooligosaccharides (LOS) (p < 0.01), whereas no significant difference in proteolytic activity was observed among the three strains (p = 0.192). LpM exhibited significantly higher water-holding capacity and cell-surface hydrophobicity than EhM and EfM (p < 0.01), along with a higher auto-aggregation rate (p < 0.01). Conversely, Eh and Ef displayed stronger co-aggregation with Staphylococcus aureus, Escherichia coli, and Salmonella, as well as greater antibacterial activity, with Ef being the most effective (p < 0.01). All three strains were highly sensitive to kanamycin, streptomycin, tetracycline, and doxycycline, whereas Eh and Ef showed intermediate sensitivity or resistance to several antibiotics. After fermentation, LpM and EhM retained IgG more effectively, while EfM consumed the least lactose. EhM produced the most acetic acid, LpM was dominant in propionic and butyric acid production, and both LpM and EhM generated more lactic acid than EfM (p < 0.01). The fermented milks displayed distinct 2,2-Diphenyl-1-picrylhydrazyl(DPPH) and hydroxyl-radical-scavenging capacities; LpM possessed the strongest antioxidant activity, followed by EhM, while EfM showed the weakest antioxidant performance (p < 0.01). During in vitro fermentation, both LpM and EhM produced more SCFAs than EfM. Through 16S rRNA gene sequencing it was revealed that LpM increased the abundance of Butyricicoccus pullicaecorum, Lactococcus, Streptococcaceae, and Lachnospiraceae. Although Eh and Ef could enrich genera such as Bacillus to inhibit diarrhea-associated pathogens, they also coincided with an increase in opportunistic pathogens including Escherichia-Shigella, Klebsiella, and Enterococcus. Therefore, Lp represents a promising candidate strain of bovine-derived lactic acid bacteria for fermented milk aimed at preventing calf diarrhea. Notably, Eh and Ef also enriched opportunistic pathogens, and further safety assessment is required before practical application.

Culled beef cows may retain practical value for fattening after reproductive use, but the rumen and host physiological characteristics associated with this response remain incompletely characterized. This study compared culled beef cows (CBC; n = 9) with fattening bulls (CON; n = 9) using 18 healthy Simmental crossbred cattle fed the same experimental total mixed ration (TMR) under identical feedlot conditions. Growth performance, body condition score (BCS), apparent nutrient digestibility, economic outcomes, blood indices, rumen fermentation, ruminal enzyme activities, rumen metagenomes, and plasma untargeted metabolomes were assessed. Compared with CON, CBC had higher final body weight, average daily gain, feed intake, and apparent digestibility of dry matter, crude protein, and neutral detergent fiber, whereas the feed-to-gain ratio did not differ significantly between groups (p = 0.692). BCS increased significantly from day 0 to day 60 within both cattle categories. Because different, non-cross-calibrated BCS reference frameworks were used, no between-group comparison of BCS was made. Economic analysis indicated that the value attributable to actual live-weight gain was similar between groups when within-category prices were held constant, whereas the relative economic outcome varied with feed costs and changes in live-cattle market prices. CBC also showed more propionate-oriented fermentation, greater fibrolytic enzyme activities, distinct carbohydrate-active enzyme profiles, higher insulin and leptin, and lower non-esterified fatty acids and β-hydroxybutyrate. Because lean and fat mass were not directly measured, the tissue basis of the greater weight gain remains unresolved. Overall, CBC exhibited a stronger fattening response accompanied by coordinated differences in rumen function and host metabolic profiles.

Mongolian cattle exhibit exceptional roughage tolerance due to their rumen microbiome’s robust fiber-degrading capacity, enabling efficient utilization of low-quality forage under the Mongolian Plateau’s seasonal fluctuations. This study compared rumen microbial composition, CAZyme profiles, fermentation parameters, and metabolic pathways in cattle fed fresh grass (FG) versus hay to elucidate microbe-metabolite interactions underlying fiber digestion. Thirty non-pregnant female Mongolian cattle (460 ± 35 kg, 3–4 years old) were randomly divided into two groups ( n = 15/group): one grazed on FG, the other housed and fed autumn-harvested hay (HG). Six animals per group were subsampled for rumen fluid collection and multi-omics analyses ( n = 6/group, total n = 12). Compared with the FG group, the HG group showed an increased molar proportion of acetate and a higher acetate-to-propionate ratio, along with reduced molar proportions of propionate and butyrate in rumen fermentation parameters. Metagenomic analysis revealed a higher abundance of Bacteroidales bacteria and anaerobic fungi (including Neocallimastix sp. JGI-2020a and Piromyces sp. E2) in the HG group. Functional annotation further indicated enriched carbohydrate metabolism pathways in the HG group, along with a greater diversity of CAZymes, particularly those involved in hemicellulose and pectin degradation. Metabolomics identified 13 differentially abundant carbohydrate metabolites, with gluconolactone upregulated in the HG group. Additionally, carbohydrate metabolism pathways identified in the metabolome corroborated the reliability of the metagenomic functional annotations. Correlation network analysis revealed positive associations of Bacteroidaceae bacteria, Neocallimastix sp. JGI-2020a, and Piromyces sp. E2 with acetate, hemicellulose-degrading GHs, and carbohydrate metabolic pathways. In conclusion, hay feeding enhanced ruminal fiber degradation in Mongolian cattle through increased Bacteroidales and anaerobic fungi, diversified CAZymes (especially hemicellulases/pectinases), and upregulated carbohydrate metabolism, reflecting microbial adaptation to low-quality forage. IMPORTANCE: Mongolian cattle’s superior roughage tolerance depends on a specialized rumen microbiome that degrades fibrous substrates via diverse CAZymes. However, microbe-metabolite interactions driving fiber digestion in this breed remain poorly understood. This study revealed an increased abundance of bacteria and fungi involved in rumen fiber degradation, which may be responsible for secreting enzymes associated with hemicellulose and pectin breakdown. Furthermore, the upregulation of key metabolites, including gluconolactone, indirectly promotes acetate production through pathways such as glycolysis and the pentose phosphate pathway. These findings reveal microbial adaptations enhancing low-quality forage utilization, offering new strategies for improving ruminant efficiency in seasonal or resource-limited grazing systems.

Oat is a forage with high protein value (10–14% DM) and good palatability, and is considered one of the main feed sources for ruminants. In this experiment, Lacticaseibacillus rhamnosus and Lentilactobacillus buchneri were selected as silage additives to investigate the fermentation quality, nutrient composition, microbial community and relationship between fermentation products and bacterial community of small-bale oat silage after ensiling. The experiment was set up with three treatment groups and three replications in each group, which were the control (C) group, L. rhamnosus (LR) group and L. buchneri (LB) group, and oat silages were subjected to 10-day and 30-day storage periods. The results show that both LR and LB additions significantly increased water-soluble carbohydrate, crude protein, lactic acid, propionic acid and acetic acid contents, and decreased pH, butyric acid, acid detergent fiber, neutral detergent fiber, and ammonia nitrogen contents and yeast and enterobacteria numbers in small-bale oat silage, compared with the C group. The highest content of acetic acid and the lowest numbers of enterobacteria and yeast were found in the LB group after 30 days of fermentation. Lentilactobacillus and Lacticaseibacillus were the dominant genera in the LB and LR groups, regardless of fermentation time. Lentilactobacillus and Lacticaseibacillus were positively correlated with a correlation value of 0.9, but both were negatively correlated with Bacillus. Lentilactobacillus and Lacticaseibacillus were positively correlated with acetic and lactic acids, while pH and butyric acid were positively correlated with Bacillus. This experiment revealed that the addition of homofermentative and heterofermentative lactic acid bacteria enhanced the relative abundance of Lentilactobacillus and Lacticaseibacillus, reduced harmful microbes, and improved fermentation quality of small-bale oat silage.

Agricultural wastes such as sugar beet byproducts and corncobs face challenges including high fiber content and low microbe–substrate interaction efficiency during their storage and conversion into animal feed resources. This study evaluated the effects of Lentilactobacillus buchneri and cellulase supplementation on fermentation quality, microbial community structure, and the in vitro fermentation rate of mixed silage containing sugar beet tops and corncobs (air-dried). Sugar beet tops and corncobs were mixed at a fresh weight ratio of 9:1 and divided into three treatments—no additives (CK), Lentilactobacillus buchneri (LB, 1 × 10⁶ CFU·g⁻¹ Lentilactobacillus buchneri), Lentilactobacillus buchneri and cellulase (LBC, 1 × 10⁶ CFU·g⁻¹ Lentilactobacillus buchneri and 0.1 g kg⁻¹ cellulase)—and subjected to anaerobic fermentation for 60 days. The results showed that LB and LBC treatments reduced the losses of crude protein (CP) and water-soluble carbohydrate (WSC) (p < 0.05) and decreased the contents of neutral detergent fiber (NDF) and acid detergent fiber (ADF) (p < 0.05). Furthermore, LB and LBC treatments significantly increased the yields of lactic acid (by 31% and 46%, respectively) and acetic acid (by 60% and 78%, respectively) after anaerobic fermentation. Microbial community analysis revealed that Lactiplantibacillus (79~85%) was the dominant genus in both LB and LBC treatments, followed by Levilactobacillus (9~15%); however, principal coordinate analysis (PcoA) showed significant differences in bacterial communities between the LB and LBC treatment. The LBC treatment significantly enriched Levilactobacillus, which exhibited significant positive or negative correlations with multiple fermentation indicators. In addition, in vitro fermentation trial demonstrated that the silage treated with LBC showed higher in vitro dry matter digestibility (IVDMD) and better fermentation characteristics during in vitro fermentation (p < 0.05), with significantly increased total volatile fatty acids (TVFA) and butyric acid (BA) contents, and a decreased acetic acid content (p < 0.05). During in vitro fermentation, the LBC treatment had higher total gas production, as well as lower methane and carbon dioxide emissions (p < 0.05). Under the synergistic effect of Lentilactobacillus buchneri and cellulase, the fermentation quality and microbial community of sugar beet top–corncob silage are improved, thereby enhancing in vitro fermentation characteristics and providing insights for the recycling of agricultural wastes.

Rumen microbiota is crucial for cellulose degradation and nutrient metabolism in ruminants. Different feeding systems like grazing and housed feeding can significantly impact it. Mongolian cattle show unique cellulose degradation ability, but functional changes under different feeding conditions are unclear. This study aims to investigate the effects of grazing and housed feeding on rumen microbiota, cellulose degradation, and metabolism in Mongolian cattle. In a 90-day trial, 12 female Mongolian cattle were divided into grazing (F group) and housed feeding (S group). Rumen samples were collected to analyze fermentation parameters, enzyme activities, microbiomes, and metabolomes. The F group had higher acetate, cellulase, xylanase, and β-glucosidase activities (p < 0.05). Bacteroidota and Prevotella were more abundant (p < 0.05), while Firmicutes and Ruminococcus were less abundant (p < 0.05) in the F group. Carbohydrate metabolic pathways and CAZymes (GH2, GH10) were upregulated in the F group, while the S group had enriched purine metabolic pathways and CAZyme (GH31). A total of 64 differential metabolites were found, with subaphylline upregulated in the F group and L-arogenate in the S group (p < 0.05). Grazing increased cellulose degradation and subaphylline production in Mongolian cattle, while housed feeding improved starch utilization efficiency and fat synthesis. These findings provide a basis for optimizing feeding strategies and improving fibrous feed resource utilization in Mongolian cattle.

Objective: This study aimed to assess the impact of corn straw-based unfermented and fermented total mixed rations (TMR) supplemented with exogenous cellulase on the in vitro fermentation characteristics, growth performance, feeding behavior, apparent digestibility, rumen fermentation and digestive enzyme activities of Chinese Simmental bulls.Methods: Unfermented (direct spraying of exogenous cellulase onto TMR, TMR) and fermented (exogenous cellulase fermentation for more than 7 d, fermented total mixed rations [FTMR]) TMR were collected, dried, powdered and used as fermentation substrates. The fermentation liquid was ruminal fluid collected from Chinese Simmental bulls. The artificial rumen culture fluid were continuously cultured in vitro for 48 h. Based on the diets they were fed, 24 healthy Chinese Simmental bulls (average weight of 495.93±10.89 kg) were randomly divided into two groups, with 12 bulls in each group, which were fed TMR or FTMR. The study lasted 56 d.Results: In in vitro experiments, the neutral detergent fiber (NDF) degradability and total volatile fatty acid, propionate, iso-butyrate, iso-valerate and valerate concentrations were greater in the FTMR group (p<0.05) than in the TMR group. However, the methane production, pH and acetate/propionate (A/P) of the FTMR group tended to be lower (p<0.05) than those of the TMR group. In the in vivo experiments, the average daily gain, eating rate, and feed efficiency of the FTMR groups were greater (p<0.05) than those of the TMR group. Similarly, the NDF degradability of the FTMR group was greater (p<0.05) than that of the TMR group. Compared to those in the TMR group, the concentrations of total volatile fatty acids, iso-butyrate, propionate and butyrate were greater in the FTMR group (p<0.05), and the A/P ratio was lower (p<0.05). Similarly, cellulase, xylanase, and β-glucosidase activities were greater (p<0.05) in the FTMR group than in the TMR group.Conclusion: Corn straw-based FTMR supplemented with exogenous cellulase play a vital role in decreasing the structural carbohydrate content of TMR and ruminal methane production in vitro, improving nutrient digestion and absorption, optimizing rumen fermentation, and improving the growth performance of beef cattle.

Aflatoxin B1 (AFB1) is the most toxic mycotoxin and is widespread in moldy feed. The use of biological removal methods to reduce AFB1 has become a research hotspot. This study aimed to isolate lactic acid bacteria (LAB) capable of removing AFB1 from moldy feeds and assessed the removal capacity under various environmental conditions. A strain named Lactobacillus brevis DN-1 was isolated from feed samples and showed 71.38% AFB1 percent removal. Furthermore, DN-1 showed good antifungal activity against Aspergillus flavus BNCC336156 and Aspergillus parasiticus BNCC335939. The optimum growth temperature and pH of DN-1 were 37 °C and 6.0, respectively, and DN-1 grew well in the concentration range of 0–20 µg/L AFB1. Under a temperature of 20–40 °C, pH of 3.0–9.0, and anaerobic conditions, the percent removal of AFB1 was more than 60%. An analysis of the different components of DN-1 showed that cell wall adsorption was the main removal method and suggested the pathway for AFB1 removal by LAB. In addition, strain DN-1 was used as a biological preservative in artificially contaminated peanut and sunflower cakes, which significantly inhibited the growth of mold and production of AFB1. In brief, this study highlights the potential use of DN-1 as a preventive agent against aflatoxicosis via strong removal capability in the application of fermented feed or food.

The mixture of whole-plant soybean and whole-plant corn silage (WPSCS) is nutrient balanced and is also a promising roughage for ruminants. However, few studies have investigated the changes in bacterial community succession in WPSCS inoculated with homofermentative and heterofermentative lactic acid bacteria (LAB) and whether WPSCS inoculated with LAB can improve fermentation quality by reducing nutrient losses. This study investigated the effect of Lactobacillus plantarum (L. plantarum) or Lactobacillus buchneri (L. buchneri) on the fermentation quality, aerobic stability, and bacterial community of WPSCS. A 40:60 ratio of whole-plant soybean corn was inoculated without (CK) or with L. plantarum (LP), L. buchneri (LB), and a mixture of LP and LB (LPB), and fermented for 14, 28, and 56 days, followed by 7 days of aerobic exposure. The 56-day silage results indicated that the dry matter content of the LP and LB groups reached 37.36 and 36.67%, respectively, which was much greater than that of the CK group (36.05%). The pH values of the LP, LB, and LPB groups were significantly lower than those of the CK group (p < 0.05). The ammoniacal nitrogen content of LB was significantly lower than that of the other three groups (p < 0.05), and the ammoniacal nitrogen content of LP and LPB was significantly lower than that of CK (p < 0.05). The acetic acid content and aerobic stability of the LB group were significantly greater than those of the CK, LP, and LPB groups (p < 0.05). High-throughput sequencing revealed a dominant bacteria shift from Proteobacteria in fresh forage to Firmicutes in silage at the phylum level. Lactobacillus remained the dominant genus in all silage. Linear discriminant analysis effect size (LEFSe) analysis identified Lactobacillus as relatively abundant in LP-treated silage and Weissella in LB-treated groups. The results of KEGG pathway analysis of the 16S rRNA gene of the silage microbial flora showed that the abundance of genes related to amino acid metabolism in the LP, LB, and LPB groups was lower than that in the CK group (p < 0.05). In conclusion, LAB application can improve the fermentation quality and nutritional value of WPSCS by regulating the succession of microbial communities and metabolic pathways during ensiling. Concurrently, the LB inoculant showed the potential to improve the aerobic stability of WPSCS.