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The influence of molasses (M) and Lactobacillus rhamnosus (LR) on fermentation products, microbial communities, and metabolites in big-bale alfalfa silage was investigated. Alfalfa (Medicago sativa L.) was harvested at the third growth stage during the flowering stage in the experimental field of Linhui Grass Company from Tongliao City, Inner Mongolia. An alfalfa sample without additives was used as a control (C). M (20 g/kg) and LR (106 cfu/g) were added either alone or in combination. Alfalfa was fermented for 7, 14, and 56 d. Lactic acid content in the M, LR, and MLR groups increased, whereas the pH value and butyric acid, 2,3-butanediol, and ethanol contents decreased compared to those of C group after 7, 14, and 56 d of fermentation. A two-way analysis of variance (ANOVA) was performed to estimate the results. The LR group exhibited increased Lactobacillus abundance, whereas the M and MLR groups showed increased Weissella abundance compared to the C group. The relative contents of amino acids (tyrosine, isoleucine, threonine, arginine, valine, and citrulline) in the M and MLR groups were higher than those in the C group. During fermentation, the M, LR, and MLR groups showed decreased phenylalanine, isoleucine, and ferulic acid contents. Amino acids such as isoleucine and L-aspartic acid were positively correlated with Lactobacillus but negatively correlated with Weissella. In conclusion, combining high-throughput sequencing and liquid chromatography–mass spectrometry during anaerobic alfalfa fermentation can reveal new microbial community compositions and metabolite profiles, supporting the application of M, LR, and MLR as feed fermentation agents.

The objective of this experiment was to investigate the effects of Lactobacillus plantarum and molasses on the nutrient composition, fermentation quality, bacterial count, aerobic stability, and microflora of alfalfa silage in sandy grasslands. The experimental treatments included control (CK), 106 CFU/g Lactobacillus plantarum (L), 5% molasses (M), and 106 CFU/g Lactobacillus plantarum + 5% molasses (LM). The nutrient composition, fermentation quality, bacterial count, aerobic stability, and microflora were determined after 14 days and 56 days of ensiling, respectively. The results showed that the addition of L, M, and LM reduced dry matter loss (DM), neutral detergent fiber (NDF), and acid detergent fiber (ADF) content, and increased water-soluble carbohydrates (WSC) and ether extract (EE) content, compared to the CK group. Meanwhile, more lactic acid (LA) and accelerated fermentation were observed, causing the pH value to drop below 4.5 in the L, M, and LM groups after 56 days of ensiling. The addition of L, M, and LM promoted lactic acid bacteria (LAB), and inhibited yeast. The addition of L significantly increased the content of acetic acid (AA). In terms of microflora, the addition of L, M, and LM made Firmicutes become the dominant bacterial phylum earlier, while Lactobacillus, Weissella, and Pediococcus had a higher abundance. According to the result of Pearson's correlation, there is a very significant negative correlation between pH value and Lactobacillus (P < 0.01) and a very significant positive correlation between pH value and Lactococcus, Enterobacter, Enterococcus, and Leuconostoc (P < 0.01), which may be inhibited by Lactobacillus under the decreased pH value. The results of the prediction of microbial genes indicated that the addition of M could enhance the carbohydrate metabolism and membrane transport metabolism, which may contribute to LA production by LAB metabolism. In general, L, M and LM all improved the fermentation quality and reduced the loss of nutrients to varying degrees, but considering the fermentation quality, the overall effects of M and LM were better than L. M and LM are recommended to be used as silage additives in the process of alfalfa silage in sandy grasslands to improve the quality.

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

3-Nitrooxypropanol (3-NOP) is a nitrooxy compound that specifically targets methyl-coenzyme M reductase (MCR), ultimately resulting in a reduction in methane production. In this study, we undertook an in vitro investigation of the effects of different dosages of 3-NOP on ruminal fermentation parameters, methane production, and the microbial community. A single-factor completely randomized design was adopted, comprising a control treatment (C), where no 3-NOP was added to the fermentation substrate, and three 3-NOP treatments, where 0.025 mg (low-dose treatment, LD), 0.05 mg (medium-dose treatment, MD), or 0.1 mg (high-dose treatment, HD) was added to 1 g of fermentation substrate (DM basis), followed by incubation for 24 h in vitro. The results showed that, compared with the control treatment, the three dosages of 3-NOP reduced total gas production, methane production, and acetate production (all p < 0.01). In contrast, 3-NOP treatment increased H2 production and the molar proportions of propionate and butyrate (all p ≤ 0.02), resulting in a decrease in the acetate-propionate ratio (p < 0.01). Meanwhile, the microbial profiles were not altered by the treatments, but the relative abundances of Prevotella, Methanobrevibacter, and Ophryoscolex were increased by the MD and HD treatments (all p < 0.01), whereas those of Methanosarcina, Methanosaeta, Sphaerochaeta, RFN20, Entodinium, and Diplodinium were decreased by the HD treatment (all p ≤ 0.03). Moreover, the results of a correlation analysis showed that there was a certain correlation between these microorganisms and total gas production, methane production, H2 production, acetate, propionate, and butyrate. In summary, under in vitro conditions, the addition of 3-NOP to the diet affected the microbial community structure, thereby altering the ruminal fermentation pattern and reducing methane production. Our results indicated that 0.05 mg per g of dietary DM is the recommended inclusion ratio for 3-NOP in the diet of lambs.

With growing restrictions on the use of antibiotics in animal feed, plant extracts are increasingly favored as natural feed additive sources. Glycyrrhiza polysaccharide (GP), known for its multifaceted biological benefits including growth promotion, immune enhancement, and antioxidative properties, has been the focus of recent studies. Yet, the effects and mechanisms of GP on broiler growth and meat quality remain to be fully elucidated. This study aimed to investigate the effects of GP on growth, serum biochemistry, meat quality, and gene expression in broilers. The broilers were divided into five groups, each consisting of five replicates with six birds. These groups were supplemented with 0, 500, 1,000, 1,500, and 2,000 mg/kg of GP in their basal diets, respectively, for a period of 42 days. The results indicated that from day 22 to day 42, and throughout the entire experimental period from day 1 to day 42, the groups receiving 1,000 and 1,500 mg/kg of GP showed a significant reduction in the feed-to-gain ratio (F:G) compared to the control group. On day 42, an increase in serum growth hormone (GH) levels was shown in groups supplemented with 1,000 mg/kg GP or higher, along with a significant linear increase in insulin-like growth factor-1 (IGF-1) concentration. Additionally, significant upregulation of GH and IGF-1 mRNA expression levels was noted in the 1,000 and 1,500 mg/kg GP groups. Furthermore, GP significantly elevated serum concentrations of alkaline phosphatase (AKP) and globulin (GLB) while reducing blood urea nitrogen (BUN) levels. In terms of meat quality, the 1,500 and 2,000 mg/kg GP groups significantly increased fiber density in pectoral muscles and reduced thiobarbituric acid (TBA) content. GP also significantly decreased cooking loss rate in both pectoral and leg muscles and the drip loss rate in leg muscles. It increased levels of linoleic acid and oleic acid, while decreasing concentrations of stearic acid, myristic acid, and docosahexaenoic acid. Finally, the study demonstrated that the 1,500 mg/kg GP group significantly enhanced the expression of myogenin (MyoG) and myogenic differentiation (MyoD) mRNA in leg muscles. Overall, the study determined that the optimal dosage of GP in broiler feed is 1,500 mg/kg.

BackgroundFreshwater snails are not only intermediate hosts for parasites but also an important part of the food chain as they convert plant biomass and humus into animal biomass. However, being widely distributed in freshwater environments, snails are highly affected by human activities, which makes their adaptation to altering environments challenging. The gut microbiome helps animals in their digestion, immune system, growth and adapting to changing environments. The effect of host species on intestinal microbial community has been poorly studied in snails.MethodsIn this study, single-molecule real-time sequencing technology (SMRT) was used to obtain full-length 16S rRNA genes to determine the intestinal microbiomes of three species of freshwater snails (SQ: Sinotaia quadrata, BU: Boreoelona ussuriensis, RP: Radix plicatula) with similar feeding habits in a same water environment.ResultsUnifrac PCoA (P

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.

Caragana korshinskii is a forage shrub species with high-protein content that has been extensively used to alleviate feed shortages for ruminants in northern China. Herein, we investigated the effects of Lactobacillus rhamnosus and Lactobacillus buchneri on the fermentation quality, aerobic stability, and microbiome composition and the predicted functional characteristics of C. korshinskii silage. C. korshinskii silages were inoculated with and without L. rhamnosus or L. buchneri. After 14 and 56 days of ensiling, the aerobic stability was determined. The results revealed that after 14 and 56 days of ensiling, L. rhamnosus- and L. buchneri-inoculated silage exhibited increased acetic acid and lactic acid contents, whereas the pH and 2,3-butanediol and butyric acid contents were decreased compared with those of the control silage. The control silages that were opened at 14 and 56 d, deteriorated during the aerobic stability test, whereas silages inoculated with L. rhamnosus and L. buchneri did not exhibit any aerobic deterioration. The control silage showed an increased Clostridium and Bacillus abundance, whereas Lactobacillus abundance decreased compared with L. rhamnosus- and L. buchneri-inoculated silages, following the 7 days of aerobic exposure. The fermentation parameters were associated with microbial communities, including Lactobacillus, Pedicoccus, Weissella, Clostridium, and Bacillus. Carbohydrate and amino acid metabolisms in the control silage decreased after 7 days of aerobic exposure compared with lactic acid bacteria-inoculated silages. To conclude, next-generation sequencing combined with 16S ribosomal RNA gene-predicted functional analyses might provide new information about the silage quality during fermentation and the aerobic stability.

In the present study, we investigated the effects of cellulase and Lactiplantibacillus plantarum (L. plantarum) on the fermentation quality, microbial diversity, gene function prediction, and in vitro rumen fermentation parameters of the Caragana korshinskii (C. korshinskii) silage. The experimental treatment groups included control (CK), cellulase (C), L. plantarum (L), and cellulase + L. plantarum (CL). Triplicate silos were sampled after 14 and 56 days of ensiling. The microbial diversity of C. korshinskii silage at 56 days was analyzed using Illumina MiSeq sequencing technology, and the effects of C. korshinskii silage on rumen fermentation were evaluated by the in vitro artificial rumen gas production method. The results showed that the addition of cellulose and L. plantarum treatments reduced ammonia-nitrogen (NH3-N), dry matter (DM), neutral detergent fiber (NDF), and acid detergent fiber content during ensiling. Compared with the CK group, higher lactic acid content was observed in the silage of the C and L groups, but the CL group had a higher acetic acid (AA) content. Compared with the CK group, the relative abundance of Lactiplantibacillus significantly increased, but that of Enterococcus, Weissella, Enterobacter, and Pediococcus significantly decreased in all other groups except the C group (p < 0.05). The results of gene function prediction were as follows: pyruvate kinase, 1-phosphofructokinase, and fructokinase were lactate production enzymes, which suggested the association of the high AA content in the CL group to the high abundance of 6-phosphate glucose dehydrogenase. The abundance of H + -transporting ATPase and ATP-binding cassette (ABC) transporters in the L and CL groups was higher than that in the CK groups. Metabolism of terpenoids and polyketides, mismatch repair, DNA replication, nucleotide excision repair, and homologous recombination increased in the CL group compared with those in the CK group. Compared with the CK group, NH3-N and microbial protein content and the degradation rates of DM and NDF increased in the L and CL groups. To conclude, the L and CL groups had increased Lactiplantibacillus abundance, improved fermentation quality, and high DM degradation rates compared with the CK group.

Leymus chinensis is an important crop that can be fed to ruminants. The purpose of this study was to investigate the roles of Lactiplantibacillus plantarum and Lentilactobacillus buchneri in fermentation quality, aerobic stability, and dynamics of wilted L. chinensis silage microorganisms. Wilted L. chinensis silages were ensiled with/without L. plantarum and L. buchneri. After 14 and 56 days of ensiling, the silos were opened and subjected to a 7-day aerobic deterioration test. This study looked at the composition of fermentation products as well as the microbial communities in silage. Silage inoculated with L. plantarum and L. buchneri had an increased lactic acid content as well as lactic acid bacterial (LAB) quantity, but a decrease in pH and levels of butyric acid, 2,3-butanediol, and ethanol was observed during ensiling. Non-treated and L. plantarum-treated silages deteriorated in the 7-day spoilage test after opening day-14 silos, whereas L. buchneri-inoculated silage showed no signs of deterioration. Lactobacillus abundance increased in the 7-day spoilage test after opening day-56 silos, while undesirable microorganisms such as Acetobacter, Bacillus, and molds, namely, Aspergillus and Penicillium were inhibited within L. plantarum- and L. buchneri-inoculated silages. The composition of fermentation products was related to the bacterial community, particularly Lactobacillus, Enterococcus, and Acetobacter. To summarize, L. plantarum- and L. buchneri-inoculated silage enhanced fermentation quality during ensiling and inhibited aerobic spoilage in a 7-day spoilage test of 56 days ensiling within wilted L. chinensis silage.

Alfalfa is conserved in silo-type bunkers in the cold and humid regions of Inner Mongolia, China. Its quality is essential to ensure a healthy and sustainable dairy production. However, the impact of environmental factors on the microbiota and fermentation products of alfalfa silage remains unclear. The present research examined changes in the microbiota and fermentation products and their association with environmental parameters in 72 samples collected from 12 farms located at 4 different latitudes and longitudes across four regions. The samples were labeled with distinct codes, A, B, and C, from the cold–rainy region, D, E, and F, from the warm–rainy region, G, H, and I from the cold–dry region, and J, K, and L from the warm–dry region. The lactic acid levels ranged from 14.25 to 24.27 g/kg of DM across all samples. The pH and concentrations of NH3-N and butyric acid in samples A, B, and H were higher (p < 0.01) than in the other samples. Samples D and E had higher acetic acid concentrations and 1, 2-propanediol content (p < 0.01). The fresh material was dominated by Pantoea and Pseudomonas, whereas Lactobacillus was the most dominant genus in all silages, except for the B silage. The A, B, and H silages contained more Clostridium but less Lactobacillus than the other silages. The lactic acid levels were strongly associated with Lactobacillus plantarum, Weissella paramesenteroides, Lactobacillus acetotolerans, Pedobacter borvungensis, and Lactobacillus brevis (p < 0.01). In contrast, the pH and the NH3-N and butyric acid concentrations were strongly associated (p < 0.01) with the presence of Clostridium estertheticum. A correlation analysis revealed that precipitation, temperature, longitude, and latitude were the most critical factors influencing epiphytic microbes in the fresh material. After silage fermentation, low-temperature conditions significantly affected the fermentation products and microbial community composition. In conclusion, the microbial community of silages is distinctive in cold and humid regions, and climatic parameters ultimately affect the microbiota and fermentation products. Furthermore, the findings of this study demonstrate that Illumina MiSeq sequencing combined with environmental factor assessment might provide new information about the microbiota composition and fermentation quality of silages, facilitating the achievement of high-quality silage.