Comparison of the effects of rumen-protected and unprotected L-leucine on fermentation parameters, bacterial composition, and amino acids metabolism in in vitro rumen batch cultures.

This study was conducted to compare the effects of rumen-protected (RP-Leu) and unprotected L-leucine (RU-Leu) on the fermentation parameters, bacterial composition, and amino acid metabolism in vitro rumen batch incubation. The 5.00 g RP-Leu or RU-Leu products were incubated in situ in the rumen of four beef cattle (Bos taurus) and removed after 0, 2, 4, 6, 12, 16, and 24 h to determine the rumen protection rate. In in vitro incubation, both RP-Leu and RU-Leu were supplemented 1.5 mmol/bottle (L-leucine HCl), and incubated after 0, 2, 4, 6, 8, 12, and 16 h to measure gas production (GP), nutrient degradability, fermentation parameters, bacterial composition, and amino acids metabolism. Results from both in vitro and in situ experiments confirmed that the rumen protection rate was greater (p < 0.01) in RP-Leu than in RU-Leu, whereas the latter was slow (p < 0.05) degraded within incubation 8 h. Free leucine from RP-Leu and RU-Leu reached a peak at incubation 6 h (p < 0.01). RU-Leu supplementation increased (p < 0.05) gas production, microbial crude protein, branched-chain AAs, propionate and branched-chain VFAs concentrations, and Shannon and Sobs index in comparison to the control and RP-Leu supplementation. RU-Leu and RP-Leu supplementation decreased (p < 0.05) the relative abundance of Bacteroidota, which Firmicutes increased (p < 0.05). Correlation analysis indicated that there are 5 bacteria at the genus level that may be positively correlated with MCP and propionate (p < 0.05). Based on the result, we found that RP-Leu was more stable than RU-Leu in rumen fluid, but RU-Leu also does not exhibit rapid degradation by ruminal microbes for a short time. The RU-Leu was more beneficial in terms of regulating rumen fermentation pattern, microbial crude protein synthesis, and branched-chain VFAs production than RP-Leu in vitro rumen conditions.

Antibacterial and antifungal activity of curcumin and methylene blue associated with laser on bacterial and fungal strains

Objective:To analyze the effect of methylene blue and 10% curcumin in fungi and bacteria through an in vitrostudy using photodynamic therapy (PDT). Methods:Curcumin and methylene blue were photosensitized by a Photon Lase III laser applied for 90 s in a dark environment within a laminar flow chamber. Enterococcus faecalisand Candida albicans strains were cultured and standardized.Then, a minimum inhibitoryconcentration (MIC) assay was conducted for these photosensitizers, with concentration variations and incubation to evaluate their antimicrobial activity. Results:With PDT, Curcumin had significant antibacterial activity against E. faecalis (MIC = 250 µg/mL).In contrast, methylene blue had antibacterial activity against E. faecalis (MIC < 12.5 µg/mL with PDT) and antifungal activity against C. albicans (MIC <12.5 µg/mL with or without PDT).Both agents showed greater efficacy in the presence of the laser.The results suggest that curcumin and methylene blue associated with laser may effectively treat microbial infections. Conclusion:Further research is needed to evaluate the efficacy and safety of using these agents in animal and human models and theireffectiveness against different bacterial and fungal strains.

Influence of carbonated acid beverage on fracture resistance and marginal gap formation in different restorative approaches to non-carious cervical lesions.

OBJECTIVE: This study is to evaluate fracture resistance, failure mode, and gap formation at the restorative interface of unrestored or restored non-carious cervical lesions (NCCLs) submitted to a short-term erosive environment. MATERIALS AND METHODS: Artificial NCCLs were produced in vitro in bovine incisors, and were randomly divided into four restorative resins groups (n = 22): nanohybrid-NR; bulk-fill-BR; flow with a nanohybrid layer-FNR; bulk-fill with a nanohybrid layer-BNR; and a group unrestored-UR (n = 16). Half of the specimens were submitted to an erosive challenge (per 5 min, 3 × a day for 7 days, before and after restoration), and the other half, was immersed in artificial saliva. After, all teeth undergone thermal (5 ºC, 37 ºC, and 55 ºC, 3600 cycles) and mechanical (50 N, 2 Hz, 300,000 cycles) aging. Eighty teeth were subjected to compressive loading, and resistance and failures were analyzed, while 24 teeth were evaluated for gaps by microcomputed tomography. Statistical tests were performed (p < 0.05). RESULTS: The restorative approaches affected fracture resistance (η2p = 0.14, p = 0.023), and gap formation (η2 = 0.18, p = 0.012) and so did the immersion medium (fracture η2p = 0.09, p = 0.008; gap η2 = 0.09, p = 0.017). BNR showed the highest resistance, while UR the lowest. FNR showed the highest gaps in both immersion media. Neither the resin groups nor the immersion media were associated with failure mode. CONCLUSIONS: The erosive immersion medium based in acid beverages has been shown to affect NCCLs with or without restoration, but when Bulk-Fill resin is covered by nanohybrid resin, the performance is good. CLINICAL RELEVANCE: Erosion negatively affects restorations, but unrestored NCCL shows worse biomechanical performance in stress-bearing situations.

Effect of slice thickness of 3D printer in fabrication of surgical guide on the accuracy of dental implant placement.

Introduction This study aimed to assess the effect of slice thickness of 3D printer in fabrication of surgical guide (SG) on the accuracy of dental implant placement. Materials and Methods After preparation of two dry human mandibles, Cone-beam computed tomography (CBCT) scans were obtained, and the location and direction of implants in the edentulous ridge of the mandible were identified using Romexis software. Data in STL format were transferred to a 3D printer and two SGs with 50 µm and 100 µm thicknesses were fabricated for each mandible. Drilling was performed using two SGs. The pre- and post-intervention CBCT scans were superimposed to measure the magnitude of differences. The two groups were compared using the Mann-Whitney U test. Results No significant difference was noted in SGs with 50 and 100 µm thicknesses in the coronal and apical regions of the implants or the depth of drilling. However, the difference in angular deviation was statistically significant. Conclusion Considering the higher accuracy of SGs with 50 µm thickness (despite the presence of a significant difference in AD between the SGs with different thicknesses), it is recommended to use a SG with 50 µm thickness in cases where anatomical limitations exist.

Effect of dietary supplementation with zinc-methionine on ruminal enzyme activities, fermentation characteristics, methane production, and nutrient digestibility: An in vitro study.

Objective: The principal objective of this research was to examine the influence of zinc-methionine (Zn-Met) addition on feed on the in vitro ruminal enzyme activities, fermentation characteristics, methane production, and digestibilities of feed nutrients. Materials and Methods: The dosage of Zn-Met as a source of organic Zn was added to feed based on dry matter (DM) as follows: 0-without Zn addition (CON), 30 Zn mg/kg-low (LZM), 60 Zn mg/kg-medium (MZM), and 90 Zn mg/kg-high (HZM). Results: The results indicated significant impacts of Zn-Met addition on various parameters. Compared to the CON group, all Zn-Met treatments resulted in increased total volatile fatty acids (VFA) (linear; p < 0.001), carboxymethyl-cellulase activity (linear; p < 0.001), and gas production at 48-h of incubation (linear; p < 0.001, quadratic; p < 0.001). Additionally, the MZM and HZM groups exhibited higher levels of amylase activity (linear; p < 0.001), protease activity (linear; p = 0.006), microbial protein (linear; p = 0.009), DM digestibility (linear; p < 0.001), organic matter (OM) digestibility (linear; p < 0.001), crude protein (CP) digestibility (linear; p = 0.004), and crude fiber (CF) digestibility (linear; p = 0.003) than CON treatment. However, the treatments did not have any noteworthy effects on pH, the individual VFA (acetate, propionate, and butyrate) proportions, NH3-N concentration, and methane production (p > 0.05). Conclusion: It could be summarized that supplementing 60 and 90 Zn mg/kg DM as Zn-Met could improve the in vitro ruminal enzyme activities, fermentation characteristics, and nutrient digestibility without affecting methane production.

A modified hydrostatic microfluidic pumpless device for in vitro murine ovarian tissue culture as research model for fertility preservation.

This study aimed to compare the efficacies of conventional and non-conventional (modified hydrostatic microfluidic pumpless device, MHPD) systems on ovarian tissue culture and in vitro follicle growth using a mouse model. A total of 56 ovarian cortical tissues retrieved from seven wild-type mice were divided into three groups: 1) fresh control, 2) conventional culture system (control), and 3) non-conventional system with MHPD. Ovarian tissues were cultured for 96 hours and evaluated for follicle morphology, developmental stage, intact follicle density, and relative gene expression levels (proliferating cell nuclear antigen, insulin like growth factor 1, BAX, and Bcl-2). Our major data demonstrated that the mean percentage of primary follicle development was increased by the MHPD (P<0.05). In addition, this device could maintain and support follicle development better than the conventional culture systems. However, the overall outcomes were not significantly improved by our first-design prototype. Consequently, nextgeneration platforms should be developed as alternative medical tools for fertility preservation research.

Does the type of sagittal split ramus osteotomy influence fixation strength? Evaluation of the mechanical behavior of two types of fixation used in three types of sagittal split ramus osteotomy.

PURPOSE: This study compared the mechanical behavior of two fixation techniques used in three sections representing the sagittal split ramus osteotomy (SSRO) in polyurethane replicas that were divided into groups, according to type of section, and sub-groups according to type of fixation, simulating 11-mm advancement and 6º clockwise mandibular rotation. METHODS: Loads were applied in two regions, aiming at progressive application and consequent strength value, measured in kilogram-force in displacements of 1, 3, 5, and 7 mm, from the load application tip. Shapiro-Wilk test was performed, followed by two-way analysis of variance (ANOVA-2 way), and Bonferroni's multiple comparison. RESULTS: The results showed no statistically significant difference in the type of section and type of fixation used when load was applied to the inter-incisor region. However, when load was applied to the first molar region, statistically significant difference was observed in 1-mm displacement, in which section described by Epker with two modifications showed greater strength, regardless of type of fixation used (p = 0.007). CONCLUSION: In the application of load in the inter-incisor region, there was no statistical difference between the type of osteotomy and the type of fixation used. When applying loads to molars, there was a difference for the type of osteotomy, where the Epker osteotomy with 2 modifications presented greater resistance, regardless of the type of fixation used.

From 3D printing to 3D bioprinting: the material properties of polymeric material and its derived bioink for achieving tissue specific architectures.

The application of 3D printing technologies fields for biological tissues, organs, and cells in the context of medical and biotechnology applications requires a significant amount of innovation in a narrow printability range. 3D bioprinting is one such way of addressing critical design challenges in tissue engineering. In a more general sense, 3D printing has become essential in customized implant designing, faithful reproduction of microenvironmental niches, sustainable development of implants, in the capacity to address issues of effective cellular integration, and long-term stability of the cellular constructs in tissue engineering. This review covers various aspects of 3D bioprinting, describes the current state-of-the-art solutions for all aforementioned critical issues, and includes various illustrative representations of technologies supporting the development of phases of 3D bioprinting. It also demonstrates several bio-inks and their properties crucial for being used for 3D printing applications. The review focus on bringing together different examples and current trends in tissue engineering applications, including bone, cartilage, muscles, neuron, skin, esophagus, trachea, tympanic membrane, cornea, blood vessel, immune system, and tumor models utilizing 3D printing technology and to provide an outlook of the future potentials and barriers.

Can monodisperse microbubble-based three-dimensional contrast-enhanced ultrasound reduce quantitative heterogeneity? An in vitro study.

BACKGROUND: Heterogeneity within the tumor may cause large heterogeneity in quantitative perfusion parameters. Three-dimensional contrast-enhanced ultrasound (3D-CEUS) can show the spatial relationship of vascular structure after post-acquisition reconstruction and monodisperse bubbles can resonate the ultrasound pulse, resulting in the increase in sensitivity of CEUS imaging. OBJECTIVES: To evaluate whether the combination of 3D-CEUS and monodisperse microbubbles could reduce the heterogeneity of quantitative CEUS. MATERIAL AND METHODS: Three in vitro perfusion models with perfusion volume ratio of 1:2:4 were set up. Both quantitative 2D-CEUS and 3D-CEUS were used to acquire peak intensity (PI) with 2 kinds of ultrasound agents. One was a new kind of monodisperse bubbles produced in this study, named Octafluoropropane-loaded cerasomal microbubbles (OC-MBs), the other was SonoVue®. The coefficient of variation (CV) was calculated to evaluate the cross-sectional variability. Pearson's correlation analysis was used to assess the correlation between weighted PIs (average of PIs of 3 different planes) and perfusion ratios. RESULTS: The average CVs of quantitative 3D-CEUS was slightly lower than that of 2D-CEUS (0.41 ±0.17 compared to 0.55 ±0.26, p = 0.3592). As for quantitative 3D-CEUS, the PI of the OC-MBs has shown better stability than that of SonoVue®, but without a significant difference (average CVs: 0.32 ±0.19 compared to 0.50 ±0.10, p = 0.0711). In the 2D-CEUS condition, the average CVs of OC-MBs group and SonoVue® group were 0.68 ±0.15 and 0.41 ±0.17 (p = 0.2747). As for 3D-CEUS condition, using OC-MBs group and SonoVue®, the r-values of the weighted PI and perfusion ratio were 0.8685 and 0.5643, respectively, while that of 2D-CEUS condition were 0.7760 and 0.3513, respectively. CONCLUSIONS: Our in vitro experiments showed that OC-MBs have the potential in acquiring more stable quantitative CEUS value, as compared to the SonoVue® in 3D-CEUS condition. The combination of 3D-CEUS and OC-MBs can reflect perfusion volume more precisely and may be a potential way to reduce quantitative heterogeneity.

Box-Wilson Design for Optimization of in vitro Levan Production and Levan Application as Antioxidant and Antibacterial Agents.

Background: Levan or fructan, a polysaccharide of fructose, is widely used in various commercial industries. Levan could be produced by many organisms, including plants and bacteria. The cloning of the gene from Bacillus licheniformis, which expressed levansucrase in Escherichia coli host, was carried out successfully. In the present study, we performed the in vitro production of levan and analyzed its potential application as antibacterial and antioxidant agents. Methods: In vitro levan production catalyzed by heterologous-expressed levansucrase Lsbl-bk1 and Lsbl-bk2 was optimized with Box-Wilson design. The antibacterial activity of the produced levan was carried out using agar well diffusion method, while its antioxidant activity was tested by free radical scavenging assays. Results: The optimum conditions for levan production were observed at 36 °C and pH 7 in 12% (w/v) sucrose for levansucrase Lsbl-bk1, while the optimum catalysis of levansucrase Lsbl-bk2 was obtained at 32 oC and pH 8 in the same sucrose concentration. The in vitro synthesized levan showed an antibacterial activity within a concentration range of 10-20% (w/v) against Staphylococcus aureus, E. coli, and Pseudomonas aeruginosa. The same levan was also able to inhibit the 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity with the antioxidant strength of 75% compared to ascorbic acid inhibition. Conclusion: Our study, therefore, shows that the optimized heterologous expression of levansucrases encoded by Lsbl-bk1 and Lsbl-bk2 could open the way for industrial levan production as an antibacterial and antioxidant agent.

A systematic review on thyroid organoid models: time-trend and its achievements.

Current in vitro models have played important roles in improving knowledge and understanding of cellular and molecular biology, but cannot exactly recapitulate the physiology of human tissues such as thyroid. In this article, we conducted a systematic review to present scientific and methodological time-trends of the reconstruction and generation of 3 D functional thyroid follicles and organoids for thyroid research in health and disease. "Web of Science (ISI)", "Scopus", "Embase", "Cochrane Library", and "PubMed" were systematically searched for papers published since 1950 to May 2020 in English language, using the predefined keywords. 212 articles were reviewed and finally 28 papers that met the inclusion and exclusion criteria were selected. Among the evidence for the examination of 3 D cell culture methods in thyroid research, there were only a few studies related to the organoid technology and its potential applications in understanding morphological, histological, and physiological characteristics of the thyroid gland and reconstructing this tissue. Besides, there was no study using organoids to investigate the tumorigenesis process of thyroid. Based on the results of this study, despite all the limitations and controversies, the exciting and promising organoid technology offers researchers a wide range of potential applications for more accurate modeling of thyroid in health and diseases and provides an excellent preclinical in vitro platform. In future, organoid technology can provide a better understanding of the molecular mechanisms of pathogenesis and tumorigenesis of thyroid tissue and more effective treatment for related disorders due to more accurate simulation of the thyroid physiology.

BMP4 micro-immunotherapy increases collagen deposition and reduces PGE2 release in human gingival fibroblasts and increases tissue viability of engineered 3D gingiva under inflammatory conditions.

BACKGROUND: We aimed to evaluate the effect of low doses (LD) bone morphogenetic protein-2 (BMP2) and BMP4 micro-immunotherapy (MI) in two in vitro models of periodontal wound healing/regeneration. METHODS: We first evaluated the effect of LD of BMP2 and BMP4 MI on a 2D cell culture using human gingival fibroblasts (hGF) under inflammatory conditions induced by IL1ß. Biocompatibility, inflammatory response (Prostaglandin E2 (PGE2) release), collagen deposition and release of extracellular matrix (ECM) organization-related enzymes (matrix metalloproteinase-1 (MMP1) and metalloproteinase inhibitor 1 (TIMP1)) were evaluated after short (3 days) and long-term (24 days) treatment with BMP2 or BMP4 MI. Then, given the results obtained in the 2D cell culture, LD BMP4 MI treatment was evaluated in a 3D cell culture model of human tissue equivalent of gingiva (GTE) under the same inflammatory stimulus, evaluating the biocompatibility, inflammatory response and effect on MMP1 and TIMP1 release. RESULTS: LD BMP4 was able to decrease the release of the inflammatory mediator PGE2 and completely re-establish the impaired collagen metabolism induced by IL1ß treatment. In the 3D model, LD BMP4 treatment improved tissue viability compared with the vehicle, with similar levels to 3D tissues without inflammation. No significant effects were observed on PGE2 levels nor MMP1/TIMP1 ratio after LD BMP4 treatment, although a tendency to decrease PGE2 levels was observed after 3 days. CONCLUSIONS: LD BMP4 MI treatment shows anti-inflammatory and regenerative properties on hGF, and improved viability of 3D gingiva under inflammatory conditions. LD BMP4 MI treatment could be used on primary prevention or maintenance care of periodontitis.

Shaping ability of hand, rotary and reciprocating files in primary teeth: a micro-CT study in vitro.

PURPOSE: Compare canal transportation and dentin removal using micro computed tomography (micro-CT) and measure instrumentation time in the root canal preparation of primary molars using K-files, ProDesign Logic and Reciproc files. METHODS: A representative sample of 81 root canals of primary molars was randomised into three groups: K-files (#15-40), ProDesign Logic (#25) and Reciproc (R25). A single-trained operator prepared all root canals. A standardised micro-CT evaluation was performed before and after instrumentation using the CTan® program. Instrumentation time was also recorded. Statistical analysis involved one-way ANOVA, Tukey's post hoc test, Kruskal-Wallis test and Dunn's test, considering a 5% level of significance. RESULTS: Reciproc led to more canal transportation than K-files and ProDesign Logic (p < 0.001) in middle third, whereas K-files led to the most canal transportation in the apical third (p = 0.017). ProDesign Logic and Reciproc removed less dentin on the opposite side of the root curvature in the apical third compared to K-files (p = 0.008). Instrumentation time was better with ProDesign Logic and Reciproc than K-files (p < 0.001). CONCLUSION: ProDesign Logic demonstrated good results with regard to canal transportation and required less instrumentation time in primary molars. Reciproc and K-files led to the most canal transportation in the middle and apical third, respectively.

In vitro Inoculation of Fresh or Frozen Rumen Fluid Distinguishes Contrasting Microbial Communities and Fermentation Induced by Increasing Forage to Concentrate Ratio.

In vitro rumen batch culture is a technology to simulate rumen fermentation by inoculating microorganisms from rumen fluids. Although inocula (INO) are commonly derived from fresh rumen fluids, frozen rumen fluids are also employed for the advantages of storing, transporting, and preserving rumen microorganisms. The effects of frozen INO on microbial fermentation and community may be interfered with by substrate type, which has not been reported. This study was designed to test whether rumen fluid treatments (i.e., fresh and frozen) could interact with incubated substrates. A complete block design with fractional arrangement treatment was used to investigate the effects of INO (fresh or frozen rumen fluids) and concentrate-to-forage ratios (C/F, 1:4 or 1:1) on rumen fermentation and microbial community. The effects of increasing C/F were typical, including increased dry matter (DM) degradation and total volatile fatty acids (VFA) concentration (P < 0.001), and decreased acetate to propionate ratio (P = 0.01) and bacterial diversity of richness and evenness (P ≤ 0.005) with especially higher fermentative bacteria such as genus Rikenellaceae_RC, F082, Prevotella, Bacteroidales_BS11, Muribaculaceaege, and Christensenellaceae_R-7 (P ≤ 0.04). Although frozen INO decreased (P < 0.001) DM degradation and altered rumen fermentation with lower (P ≤ 0.01) acetate to propionate ratio and molar proportion of butyrate than fresh INO, typical effects of C/F were independent of INO, as indicated by insignificant INO × C/F interaction on substrate degradation, VFA profile and bacterial community (P ≥ 0.20). In summary, the effect of C/F on fermentation and bacterial diversity is not interfered with by INO type, and frozen INO can be used to distinguish the effect of starch content.

Adipose and Muscle Cell Co-Culture System: A Novel In Vitro Tool to Mimic the In Vivo Cellular Environment.

A co-culture system allows researchers to investigate the complex interactions between two cell types under various environments, such as those that promote differentiation and growth as well as those that mimic healthy and diseased states, in vitro. In this paper, we review the most common co-culture systems for myocytes and adipocytes. The in vitro techniques mimic the in vivo environment and are used to investigate the causal relationships between different cell lines. Here, we briefly discuss mono-culture and co-culture cell systems and their applicability to the study of communication between two or more cell types, including adipocytes and myocytes. Also, we provide details about the different types of co-culture systems and their applicability to the study of metabolic disease, drug development, and the role of secretory factors in cell signaling cascades. Therefore, this review provides details about the co-culture systems used to study the complex interactions between adipose and muscle cells in various environments, such as those that promote cell differentiation and growth and those used for drug development.

Establishment of an in vitro model using the rat alveolar macrophage cell line NR8383.

OBJECTIVE: To establish an in vitro model of radiation-induced lung injury using rat lung alveolar macrophages (NR8383). METHODS: Using a medical electronic linear accelerator, cells were irradiated with either 0 Gy or 6 Gy X-rays. At 6, 12, 24, 30 and 48 h, the DNA damage index (8-OHdG) and lipid damage index (MDA) were measured in the two groups. We also determined the levels of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and transforming growth factor-ß (TGF-ß). RESULTS: The levels of 8-OHdG and MDA in the 6 Gy irradiation group were higher than those in the 0 Gy group at 6, 12, 24, 30 and 48 h after irradiation. The levels reached the highest value -6 h after irradiation, and then gradually decreased. The levels of the inflammatory factors TNF-α, TGF-ß and IL-6 were higher in the 6 Gy irradiation group than those in the 0 Gy group at 6, 12, 24, 30 and 48 h after irradiation. CONCLUSION: Six Gy X-ray irradiated NR8383 cells can be used to establish an in-vitro model of radiation-induced lung injury. The levels of 8-OHdG, MDA, TNF-α, TGF-ß and IL-6 can be used as effective evaluation indicators.

Effect of applying lactic acid bacteria and cellulase on the fermentation quality, nutritive value, tannins profile and in vitro digestibility of Neolamarckia cadamba leaves silage.

This study was designed to evaluate the effect of applying lactic acid bacteria (LAB, Lactobacillus plantarum LP) (0 and 1 × 106  cfu/g fresh matter (FM)) and cellulase (0, 30, 60 and 120 U/g FM) on the ensiling characteristics of N. cadamba leaves. The experimental design was a completely randomized 2 × 4 factorial arrangement. Fermentation parameters, microbial populations, chemical compositions and in vitro digestibility of the silages were measured after 60-day fermentation. LAB inoculation decreased the pH (p < 0.05), ammonium nitrogen (NH3 -N) proportion (p = 0.08), the concentrations of crude protein (CP, p < 0.05) and condensed tannins (CT, p = 0.07), and the microbial counts (p < 0.01) of LAB and yeasts along with increased (p < 0.01) concentrations of neutral detergent fibre (NDF), acid detergent fibre (ADF), acid detergent lignin (ADL) and hemicellulose. Cellulase addition increased (p < 0.01) the microbial populations and protein fractions and decreased (p < 0.01) the concentrations of acetic acid, cell wall components, total phenols (TP), soluble tannins (ST) and CT with increasing enzyme inclusions. The interaction of LAB and cellulase showed an effect (p < 0.01) on the NH3 -N proportion, microbial counts, fibre fractions and CP. No effect (p > 0.05) was found on the dry matter recovery and digestibility of the silages with LAB or cellulase application. Besides, high lactic acid concentration and true protein ratio, low pH and NH3 -N proportion, and a few spoilage organism counts were found in all silages, indicating a successful preservation. These results infer that, with some improvement, the ensiling characteristics and chemical compositions of N. cadamba leaves silage show specific responses to the addition of LAB inoculant or cellulase.