Search Results (108)

This study investigated the interactive effects of corn silage and ramie silage on in vitro rumen fermentation characteristics, aiming to provide a scientific basis and empirical evidence for the rational incorporation of ramie into ruminant diets. Four binary substrate mixtures were formulated based on dry matter (DM) mass ratios of corn silage to ramie silage: 100:0 (CON), 60:40 (R40), 20:80 (R80), and 0:100 (R100). Rumen fluid was collected from three adult Liuyang black goats surgically fitted with permanent rumen cannulas, and a standardized 48 h in vitro batch culture assay was conducted. Results demonstrated that increasing the proportion of ramie silage significantly decreased (p < 0.05) the DM degradation rate, neutral detergent fiber (NDF) degradation rate, acid detergent fiber (ADF) degradation rate, and total gas production per gram of substrate DM. Specifically, CON and R40 exhibited significantly higher values for all four parameters than R80 and R100 (p < 0.05). Methane production was significantly reduced in all ramie-containing treatments relative to CON (p < 0.05), whereas hydrogen production increased progressively with ramie inclusion level, with CON yielding significantly less H₂ than both R80 and R100 (p < 0.05). Regarding fermentation parameters, increasing ramie proportion elevated (p < 0.05) both fermentation fluid pH and the acetate-to-propionate ratio, while total volatile fatty acid (TVFA) concentration declined linearly (p < 0.05). TVFA concentrations did not differ significantly between CON and R40, yet both were significantly greater than those in R80 and R100 (p < 0.05). Collectively, these findings indicate that ramie silage is a nutritionally valuable forage with potential as a high-quality partial replacement for conventional silages in ruminant feeding systems; however, its inclusion in corn–ramie mixed silages should not exceed 40% (on a DM basis) to maintain optimal fermentative efficiency and nutrient degradability. Full article

The present study was conducted to demonstrate the effects of Codium fragile extract on methane production using in vitro and in vivo experiments. An in vitro batch experiment was conducted to evaluate different inclusion levels of Codium fragile extract (0, 0.25, and 0.5% of substrate dry matter). Methane production significantly decreased in the 0.5% treatment (p < 0.05), whereas dry matter digestibility and total volatile fatty acid concentration were not significantly affected (p > 0.05). Based on the in vitro results, an in vivo feeding experiment was conducted using a 0.5% inclusion level of Codium fragile extract on Hanwoo steers. Methane emissions were significantly decreased by approximately 10% in steers fed Codium fragile extract (p < 0.05). In contrast, rumen fermentation characteristics, feed intake, average daily gain, and blood parameters were not significantly different between the treatments (p > 0.05). These results demonstrate that a dietary additive with 0.5% Codium fragile extract effectively reduced methane emissions without negatively affecting rumen fermentation and growth performance in Hanwoo steers. Full article

The need for new feed ingredients that could reduce methane (CH₄) emissions from dairy cattle while maintaining rumen function is essential for sustainable milk production. This study aimed to evaluate the CH₄ mitigation potential of selected microalgae and macroalgae, along with an agro-industrial by-product, using two feeding strategies, and hypothesized that lipid- and polyphenol-rich materials would reduce CH₄ production in an inclusion-dependent manner. An in vitro batch culture study (24 h) was conducted to evaluate microalgae (Euglena gracilis and Aurantiochytrium spp.), macroalgae (Undaria pinnatifida), and an agro-industrial by-product (grape marc) either as feed additives (5%) or as a partial replacement of the concentrate mixture (30%, 50%, and 70%) in a basal diet consisting of 50% Klein grass hay and 50% concentrate mixture. As a feed additive, grape marc stands out for its potential to reduce CH₄ yield by about 43.3% without adversely affecting digestibility, pH, or total volatile fatty acid concentrations. When used as feed replacements, Euglena-, Aurantiochytrium-, and grape marc-based feeds reduced CH₄ yield at the highest replacement levels (50 and 70%); however, these effects were accompanied by decreased total gas production and volatile fatty acid concentrations, indicating reduced fermentation activity. Meanwhile, at a 30% replacement level, they showed promising efficiency as alternative feeds. Overall, CH₄ mitigation depends more strongly on inclusion strategy rather than feed type. Lipid-rich microalgae showed potential as concentrate replacements up to 30%, whereas grape marc was most effective as a feed additive for reducing CH₄ emissions. Full article

Background and Objectives: Current research approaches focusing on the human gut microbiota require complex in vitro systems that could provide sufficient viability and similarity with the conditions provided by the human intestine. As critical physiological functions, such as metabolic and inflammatory modulation, are associated with gut microbiota activity, complex host–microbiota interactions represent a pivotal new direction for therapeutic and nutritional interventions. However, there are several limitations to the current development of advanced in vitro models. Materials and Methods: A systematic review was performed according to the PRISMA guidelines for data collection and interpretation. Results: This manuscript summarizes the most advanced in vitro approaches for studying the gut microbiota, including batch fermentation models, dynamic fermentation models, and state-of-the-art technologies, such as organoids and gut-on-a-chip platforms. Each model offers beneficial study backgrounds, advantages, limitations, and the capacity to replicate the physiological complexity of the intestinal environment. However, due to the increased heterogeneity of the reported models, there is an urgent need for standardization. In this way, coherent regulatory frameworks are needed to guide the development and application of in vitro models. Conclusions: By consolidating knowledge and critically addressing current challenges, this study contributes to gut microbiota research by providing a direction for ethical, precise, and high-impact scientific studies. Full article

Agro-residues and aquatic biomass are potential sustainable ruminant feeds. However, their effects on rumen digestibility, fermentation, and greenhouse gas emissions remain insufficiently characterized. This study used two in vitro batch culture experiments to evaluate crushed almonds, peanut skins, and water hyacinth as feed additives (1–10%) and grass hay replacements (25–100%). Their chemical composition varied significantly. Crushed almonds are rich in crude protein (22.47%) and ether extract (38.38%). Peanut skins contained moderate protein (13.87%) and high fiber and lignin, while water hyacinth had the lowest protein (7.27%) and highest fiber and ash fractions. All ingredients had no effect on fermentation or methane production when used as additives, whereas grass hay replacement induced marked ingredient-specific responses. Crushed almonds showed the highest in vitro dry matter digestibility (IVDMD), increased propionate, and lowered the A:P ratio; however, methane production increased by 12%. Peanut skins sharply reduced IVDMD with increasing inclusion, resulting in the strongest methane mitigation (57% reduction). Water hyacinth displayed a modestly low IVDMD and moderate methane reduction (14%). In conclusion, methane production is closely associated with substrate degradability, and each ingredient exhibits distinct functional tendencies: crushed almonds enhanced energy availability and digestibility, peanut skins showed methane mitigation potential, and water hyacinth functioned primarily as a low-fermentability grass hay substitute. Full article

Faced with the challenge of reducing food waste, transforming discarded fruit into functional ingredients useful for the food industry is a valuable solution. Ingredients from fruit such as persimmons, which are rich in indigestible carbohydrates and bioactive compounds with antiradical capacity, could positively impact on the health of certain population groups due to their potential prebiotic effect. This study aimed to select the most suitable drying conditions and milling intensity for obtaining powdered persimmon ingredients with a prebiotic-like effects observed in vitro for postmenopausal women, and to evaluate this effect by considering the stimulation of health-promoting bacterial growth and short-chain fatty acids (SCFAs) production. First, the effect of the drying method (hot air drying at 60 and 70 °C, and freeze-drying) and grinding intensity on antiradical capacity, particle size, and the release of bioactive antiradical components into the intestinal lumen after an in vitro gastrointestinal digestion was determined. Next, the effect of these conditions on the microbiota composition of postmenopausal women was preliminary assessed in a batch colonic fermentation experiment for 24 h. The results showed that the ingredient dried with air at 70 °C had the highest phenol and flavonoid content, suffered the least degradation during in vitro gastrointestinal digestion and promoted the differential growth of fiber-degrader genera. Consequently, this was the ingredient selected as the most suitable. Lastly, the impact of this ingredient on the microbiota composition of 4 postmenopausal women has been evaluated in a long-term study using the Simulator of the Human Intestinal Microbial Ecosystem (SHIME^®) coupled to high throughput sequencing. The growth stimulation of health-associated bacteria, such as Akkermansia muciniphila, Faecalibacterium prausnitzii or Phascolarctobacterium faecium, and the promotion of beneficial metabolic pathways, such as the sugar uptake-specific phosphotransferase system, sugar metabolism and propionate and isobutyrate production, were detected along 14 days of persimmon powder supplementation. A holistic framework for promoting human health while advancing environmental sustainability is represented by the combination of sustainable by-product valorization and microbiota-targeted functional food development. Full article

Strategies to suppress methanogenesis must preserve the functional integrity of the rumen microbial ecosystem. Essential oils (EOs) have emerged as promising modulators of rumen microbial function, though their responses vary widely with chemical structure and inclusion level. This study evaluated the efficacy of selected EOs using detailed in vitro fermentation assays. Nine EOs—cinnamon, lavender, garlic (GAR), lemongrass (LEG), peppermint (PPM), eucalyptus, coriander, oregano, and ginger (GIN)—were evaluated for their effects on rumen fermentation and methane (CH₄) production using a 24 h in vitro batch culture system. Eight EOs were tested at two doses (Low and High) specific to each EO, while GIN was evaluated at a single dose. All treatments were incubated in a rumen fluid–buffer mix (1:1 for fermentation parameters and 1:4 for gas and CH₄ measurements) with a 55:45 forage-to-concentrate substrate (pH 6.9). Overall treatment effects were significant for all measured fermentation parameters (p < 0.01). Most treatments reduced total gas production, CH₄ emissions, and CH₄/total gas ratios compared with the control (p < 0.05), although several responses were dose-dependent or directly divergent. Essential oils showed clear, composition-dependent responses: non-terpenoid EOs produced the strongest but also the most variable antimethanogenic effects, with GAR, particularly at the lower dose, consistently achieving the greatest CH₄ inhibition while maintaining a favorable fermentation pattern. Conversely, terpenoid-based EOs induced moderate, dose-responsive CH₄ reductions with minimal effects on overall fermentation. At the higher dose, PPM suppressed CH₄ without altering major volatile fatty acid (VFA) patterns aside from increases in valerate and branched-chain VFA, whereas LEG reduced CH₄ only when accompanied by marked fermentation depression. Monensin validated its role as an effective positive control. Overall, GAR, characterized by sulfur-based bioactives, emerged as the most effective candidate for CH₄ mitigation under the tested in vitro conditions, highlighting the importance of chemical composition and inclusion level in determining efficacy and reinforcing the need for in vivo validation. Full article

Mitigating enteric methane emissions through diet formulation remains a significant challenge in cattle nutrition. This study developed a system to evaluate the methane production potential of feeds, expressed as the effective ruminal methane production rate (eRMR, mL/g dry matter [DM]), using a discontinuous in vitro ruminal fermentation system using rumen fluid. Sixteen concentrate feeds and two forages were tested, with a reference diet (ryegrass straw:corn:corn gluten feed = 1:1:1) included in each batch to standardize conditions and account for associative effects among feeds. Test feeds were incubated with the reference diet in closed bottles under strictly anaerobic conditions. Methane and total gas production were measured at 2, 4, 6, and 24 h, and true dry matter digestibility was calculated after 6 and 24 h. For each batch, sample feed values were corrected and standardized using those of the reference diet. The eRMR value was calculated by integrating a differential equation with parameters incorporating ruminal digestion and passage dynamics. The test feed eRMR values ranged from 1.2 mL/g DM (soybean meal) to 56.7 mL/g DM (soybean hull), with the reference diet at 14.8 mL/g DM. Evaluation of feed eRMR using data from two in vivo studies demonstrated strong correlations between predicted diet-specific eRMR values and measured methane emissions from Hanwoo steers (r = 0.93 and 0.85). This system, incorporating rumen dynamics with a reduced sampling schedule, provides a precise and practical tool for predicting in vivo enteric methane production and optimizing diet formulations to mitigate methane emissions from cattle. Full article

Hypoglycin A is a plant-derived protoxin that causes atypical myopathy in equids. In atypical myopathy-affected horses, metabolomic and microbiome studies have reported alterations in metabolic markers and faecal microbiota composition, pointing to a potential disruption of microbial homeostasis. However, in vivo observations are strongly confounded by host-related factors, underscoring the need for controlled in vitro approaches. To address this, we used an in vitro static batch fermentation model simulating the equine colon to investigate the direct effects of hypoglycin A on microbiota composition and activity. Faecal inocula from healthy horses were incubated in control and hypoglycin A-treated fermenters for 48 h, with serial analyses of hypoglycin A concentration, short-chain fatty acids, and 16S rRNA gene profiles. Hypoglycin A remained stable in the nutritive medium in the absence of microbiota, confirming that its degradation in inoculated fermenters was microbiota-dependent. The results showed significant microbial-associated hypoglycin A degradation without evidence of toxic metabolite formation. The analysis of α- and β-diversity revealed both an effect of incubation time, reflecting the natural temporal dynamics of microbial communities under batch fermentation, and a specific impact of hypoglycin A exposure, with certain taxa such as Paraclostridium being affected. This study provides the first in vitro evidence that the equine microbiota contributes to hypoglycin A degradation. Full article

Ruminant in vitro methodologies use washing with neutral detergent solution (NDS) after incubation to mimic ruminant digestion, which is physiologically different compared to that of horses. Our objectives were to determine if washing feed samples with NDS before in vitro fermentation (PRE) would suppress fiber digestion versus a post-incubation wash (POST), and to compare in vitro digestibility of forage-based feed mixtures with added soluble carbohydrates (CARB), soluble protein (PROT), or soluble carbohydrates and soluble protein (C + P) to only-forage samples (CONT). Dried, ground feed mixtures sealed in ANKOM filter bags were placed in Daisy^II incubators for 48 h in a split–split-plot batch culture design. Digestibility was determined as in vitro neutral detergent fiber digestibility (IVNDFD), in vitro acid detergent fiber digestibility (IVADFD), in vitro hemicellulose digestibility (IVHD), and in vitro true digestibility (IVTD). The PRE treatment decreased IVHD for CARB versus POST (p = 0.007). Pooling all mixtures, PRE decreased IVTD (p = 0.001), IVADFD (p = 0.036), and IVHD (p = 0.001) and tended to decrease IVNDFD (p = 0.072). The CARB mixture increased IVTD versus all other mixtures (p < 0.001). Pre-washing with NDS suppressed in vitro fermentation by removing soluble carbohydrates. Without removal of soluble carbohydrates to mimic in vivo digestion, fiber digestibility is likely overestimated. Full article

Objective: This study systematically evaluated the nutritional compositions and bioactive compounds of six unconventional feed resources (Pepper residue (PR), Grape marc (MC), Pepper straw (PS), Lycium barbarum branches and leaves (LBBL), Licorice straw (LS), and Cyperus esculentus leaves (CES)). It also assessed the rumen degradability and rumen fermentation characteristics at different substitution levels through in vitro and in situ methods, to explore their potential application in sheep diets. Methods: Samples were analyzed considering nutrient composition, amino acids, polyunsaturated fatty acids (PUFAs), and bioactive compounds. In situ degradation was measured using rumen-fistulated sheep, and in vitro batch fermentation culture was conducted at varying substitution levels (0–100%) to measure gas production, pH, VFAs, NH₃-N, and microbial crude protein (MCP). Results: The six unconventional feed resources showed significant differences in nutrient composition, bioactive compounds, and fermentation performance. Crude protein (CP) ranged from 4.45% to 15.76%, with LS highest in total amino acids. LBBL contained 4.24 g/kg Lycium barbarum polysaccharides, LS had 9.24 g/kg liquiritin, GM was richest in proanthocyanidins, and PS had more capsaicin than PR. PR exhibited the highest DM degradation (74.77%, p < 0.001), followed by LS; CEL was lowest. PR and LS also had the highest CP degradation. In vitro fermentation revealed significant differences in fermentation characteristics among the six feeds. At 100% replacement, PR and LS exhibited high cumulative gas production, elevated MCP concentrations, and total VFAs of 54.41 and 64.02 mmol/L (p < 0.001), respectively. At 25% replacement, GM and CEL achieved high concentrations of VFAs and maintained MCP levels of 27.84 and 31.57 mg/dL (p < 0.001). PS reached its maximum total VFAs and MCP at 50% replacement, while LBBL reached 64.90 mmol/L total VFAs and 32.63 mg/dL MCP at 75% replacement. Conclusions: Nutrient composition and degradation kinetics varied significantly among substrates. PR had the highest DM degradability, while CEL had the lowest. PR and LS maintained stable fermentation at 100% substitution. GM and CEL were most effective at 25%; PS at 50%; and LBBL at 75% substitution levels. Full article

In our previous study, short-chain inulin and inulin neoseries oligosaccharides (SCIINOs) were produced and purified from red onion juice. This study aimed to investigate the effect of SCIINOs on changes in the bacterial composition of fecal microbiota obtained from normal weight, overweight, and obese subjects using in vitro batch fermentation. Fermentation characteristics, including changes in fecal microbiota determined by the V3–V4 region of 16S rRNA amplicon sequencing, residual SCIINO content, and the resulting organic acid profiles, were determined. The results indicate that SCIINOs were fermentable, which occurred along with a decrease in the SCIINO content and an increase in lactic, acetic, propionic, and butyric acids. The microbial composition of fecal inoculum influenced the degree of SCIINO fermentation, which was then associated with the fermentation outcomes. Alpha-diversity results revealed that fermentation with and without SCIINOs decreased species richness, evenness, and diversity. Beta-diversity results revealed that fermentation of SCIINOs using all fecal inocula negatively affected the abundance of Escherichia-Shigella and Klebsiella while positively affecting the abundance of Lactococcus. The enrichment of Lactococcus was confirmed by an independent study, indicating that two reference strains of Lactococcus lactis efficiently utilized neokestose and nystose as the major FOS constituent present in SCIINOs. Full article

Allergy is recognized as a public health problem with pandemic consequences and is estimated to affect more than 50% of Europeans in 2025. Prebiotic and probiotic food implementation has recently emerged as an alternative strategy to promote immunomodulatory beneficial effects in allergic patients. Among prebiotics, Phaeophyceae algae represent a niche of research with enormous possibilities. The present study aims to evaluate the in vitro prebiotic potential of fucoidan from Fucus vesiculosus, Macrocystis pyrifera, and Undaria pinnatifida algae, to promote the growth of Limosilactobacillus reuteri and Lacticaseibacillus rhamnosus GG as probiotic bacteria added to the formulation of a novel yogurt. Concentrations of fucoidan of 100 and 2000 µg/mL were added to reference growth media and kinetic growth curves for both microorganisms were fitted to the Gompertz equation. Optimized prebiotic conditions for fucoidan were selected to validate in vitro results by means of the formulation of a novel fermented prebiotic yogurt. Conventional yogurts (including Streptococcus thermophilus and Lactobacillus delbrueckii subs. bulgaricus) were formulated with the different fucoidans, and production batches were prepared for L. rhamnosus and L. reuteri. Increased L. reuteri and L. rhamnosus populations in 1.7–2.2 log₁₀ cycles just after 48 h of in vitro exposure were detected in fucoidan supplemented yogurt. M. pyrifera and U. pinnatifida fucoidans were the most effective ones (500 µg/mL) promoting probiotic growth in new formulated yogurts (during the complete shelf life of products, 28 days). Diet supplementation with fucoidan can be proposed as a strategy to modulate beneficial microbiota against allergy. Full article

Ruminant livestock contribute significantly to methane emissions, necessitating sustainable mitigation strategies. Essential oils (EOs) show promise for modulating ruminal fermentation, but their synergistic effects remain underexplored. Two 24 h in vitro experiments evaluated the synergistic effects of EO blends on rumen microbial fermentation. Exp. 1 screened five oils using two triad combinations. Triad 1 tested 10 combinations of thyme (THY), peppermint (PPM), and cinnamon leaf (CIN) oils. Triad 2 tested 10 combinations of anise (ANI), clove leaf (CLO), and peppermint (PPM) oils. Each blend was tested at 400 mg/L, using batch culture methods measuring: pH, ammonia-N (NH₃-N), and volatile fatty acids (VFAs). The two most effective blends, designated as T1 and T2, were selected for Exp. 2 to assess total gas and methane (CH₄) production using pressure transducer methods. All treatments were incubated in a rumen fluid–buffer mix with a 50:50 forage-to-concentrate substrate (pH 6.6). In Exp. 1, data were analyzed according to the Simplex Centroid Design using R-Studio. In Exp. 2, an analysis was conducted using the MIXED procedure in SAS. Mean comparisons were assessed through Tukey’s test. The results from Exp. 1 identified CIN+PPM (80:20) and ANI+CLO (80:20) as optimal combinations, both increasing total VFAs while reducing acetate/propionate ratios and NH₃-N concentrations. In Exp. 2, both combinations significantly reduced total gas and CH₄ productions compared to the control, with CIN+PPM achieving the greatest methane reduction (similar to monensin, the positive control). Specific essential oil combinations demonstrated synergistic effects in modulating rumen fermentation and reducing methane emissions, offering potential for sustainable livestock production. Further in vivo validation is required to optimize dosing and assess long-term effects on animal performance. Full article

The increasing prevalence of non-communicable diseases (NCDs) is strongly associated with gut microbiota (GM) imbalances and reduced short-chain fatty acid (SCFA) production, primarily driven by poor diet and microbial dysbiosis. Since SCFAs are crucial for gut health, immune regulation, and inflammation control, restoring their levels is a key therapeutic target. SCFA-acylated naringin derivatives offer a novel approach by enhancing SCFA delivery and modulating GM composition. In this study, we investigated the effects of naringin acetate and naringin propionate on SCFA production using a 24 h short-term in vitro batch fecal fermentation model with microbiota from two donors. Naringin propionate and naringin plus free propionate significantly increased propionate levels by 0.74 mM and 0.75 mM, respectively (p < 0.0001), while naringin acetate induced a smaller increase of 0.26 mM. Donor-specific reflected differences in microbial communities, yet SCFA enhancement was observed across samples. Additionally, naringin treatments stimulated the growth of beneficial polyphenol-metabolizing bacteria, including Bacteroides, Streptococcus, and Eubacterium siraeum. The strong effect of naringin propionate suggests a sustained SCFA release mediated by microbial enzymes. These preliminary results highlight the potential of SCFA-acylated flavonoids as functional dietary components to increase SCFA bioavailability and support gut health, particularly from citrus-derived co-products. Full article