Search Results (105)

The impact of donor age on Adipose-derived mesenchymal stem cell (ASC) functionality and safety remains insufficiently characterized, particularly in equine models. This study investigates the influence of age on ASCs proliferation dynamics and the expression of tumorigenic and apoptosis-related markers. Equine ASCs were isolated from juvenile (<5 years), middle-aged (5–15 years), and geriatric (>15 years) horses and assayed across multiple passages. The relative mRNA expressions of pluripotency (Oct4), tumorigenic (CA9), and apoptosis-related (Bax and Bcl 2) markers were evaluated. The Gompertz growth model, population doubling time (PDT), and tissue non-specific ALP activity also followed. The expression of pluripotency and tumorigenic markers showed passage-dependent up-regulation, raising concerns about prolonged culture expansion. Apoptotic regulation displayed a shift with aging, as evidenced by alterations in the Bax/Bcl2 ratio, suggesting compromised cell survival in older ASCs. An age-associated decline in proliferation rates was established, as evidenced by declining alkaline phosphatase (ALP) activity. These findings underscore the necessity for stringent age-based selection criteria in equine stem cell therapies and the challenges associated with using autologous stem cells for regenerative therapies in aged horses. Future research should focus on molecular interventions to mitigate age-related functional decline, ensuring the safety and efficacy of ASCs-based regenerative medicine in equine practice. Full article

This study was conducted to investigate the effects of replacing fish meal with either whole-fat or defatted krill powder on the growth, body color, immunity, and related gene expression of red–white koi carp. A total of 630 red–white koi carp with an initial body mass of 13.5 ± 0.05 g were randomly divided into seven groups with three replicates per group and 30 fish per replicate. The control group was fed a basic diet (C0). The other six diets were supplemented with different levels of whole krill meal or defatted krill meal as replacements (10% whole fat, 20% whole fat, 30% whole fat, 10% defatted, 20% defatted, and 30% defatted) in the experimental groups, named W10, W20, W30, D10, D20, and D30, respectively, for a total duration of 60 days. The growth, body color, immunity and gene expression indexes were measured in the koi after completion. The results indicate the following. (1) Compared with C0, the experimental groups of koi showed a significant increase in the specific growth rate (SGR) (p < 0.05), while the hepatosomatic index (HSI) and viscerosomatic ratio (VSI) decreased. Additionally, there was a significant increase in the relative expression level of insulin-like growth factor (IGF-1) in both the liver and muscle (p < 0.05). (2) The experimental groups of koi carp exhibited a significant increase in the carotenoid content in the scales and skin, as well as an elevated relative expression level of the tyrosinase (TYR) gene in the muscle (p < 0.05). (3) The lysozyme (LZM) and superoxide dismutase (SOD) activities were significantly increased in the experimental groups compared to C0 (p < 0.05). Additionally, the SOD activity was significantly higher in the defatted groups than in the whole-fat groups (p < 0.05). Furthermore, the liver alkaline phosphatase (AKP) activity was significantly lower in the D20 and D30 groups compared to the other five groups. (4) The expression level of LPL was significantly lower in the liver and muscle of the whole-fat group compared to the defatted group (p < 0.05). Tissue section observation revealed that the hepatocytes in the W20 and W30 groups exhibited a reduced size and an increased lipid droplet count, while the vacuolar degeneration of the hepatocytes increased in the D30 group. In conclusion, replacing fish meal with whole-fat or defatted krill powder or defatted Antarctic krill meal significantly improves the growth performance, body color, and immunity of red–white koi carp. However, excessive addition of krill meal can easily cause liver damage. The recommended replacement level for whole krill powder is 20% to 30%, while defatted krill powder should be replaced at a level of 10% to 20%. Full article

Global kelp farming is garnering growing attention for its contributions to fishery yields, environmental remediation, and carbon neutrality efforts. Kelp farming systems face escalating pressures from compounded climatic and environmental stressors. A severe outbreak disaster caused extensive kelp mortality and significant economic losses in Rongcheng, China, one of the world’s largest kelp farming areas. This study investigated the growth and physiological responses of Saccharina japonica to combined stressors involving three levels of N:P ratios (10:1 as a control; 100:1 and 500:1 to represent phosphorus deficiency stress) and two temperature/light regimes (12 °C, 90 μmol photons m⁻² s⁻¹ as a control, and 17 °C, 340 μmol photons m⁻² s⁻¹ to represent thermal and high-light stress). The results demonstrated that phosphorus deficiency significantly inhibited the relative growth rate of kelp (24% decrease), and the strongest growth inhibition in kelp was observed at the N:P ratio of 500:1 combined with thermal and high-light stress. The algal tissue was whitened due to its progressive disintegration under escalating stress, coupled with damage to its chloroplasts and nucleus ultrastructures. Phosphorus-deficiency-induced declines in photochemistry (27–56% decrease) and chlorophyll content (63% decrease) were paradoxically and transiently reversed by thermal and high-light stress, but this “false recovery” accelerated subsequent metabolic collapse (a 60–75% decrease in the growth rate and a loss of thallus integrity). Alkaline phosphatase was preferentially activated to cope with phosphorus deficiency combined with photothermal stress, while acid phosphatase was subsequently induced to provide auxiliary support. S. japonica suppressed its metabolism but upregulated its nucleotides under phosphorus deficiency; however, the energy/amino acid/coenzyme pathways were activated and a broad spectrum of metabolites were upregulated under combined stressors, indicating that S. japonica employs a dual adaptive strategy where phosphorus scarcity triggers metabolic conservation. Thermal/light stress can override phosphorus limitations by activating specific compensatory pathways. The findings of this study provide a foundation for the sustainable development of kelp farming under climate and environmental changes. Full article

Bone tissue engineering (BTE) emerged as a practical approach to tackle prosthetic industry limitations. We merge aspects from developmental biology, engineering and medicine with the aim to produce fully functional bone tissue. Mesenchymal stem cells have the capability of self-renewal and specific lineage differentiation. Herein lies their potential for BTE. Among MSCs, human dental pulp stem cells have a higher proliferation rate, shorter doubling times, lower cellular senescence, and enhanced osteogenesis than hBM-SCs under specific conditions. In addition, these cells are readily accessible and can be extracted through a subtle extraction procedure. Thus, they garner fewer moral concerns than most MSCs available and embody a promising cell source for BTE therapies able to replace hBM-MSCs. Interestingly, their study has been limited. Conversely, there is a need for their further study to harness their true value in BTE, with special emphasis in the design of bioprocesses able to produce viable, homogenous bone constructs in a clinical scale. Here, we study the osteogenic differentiation of hDPSCs encapsulated in alginate hydrogels under suspended culture in a novel perfusion bioreactor. The system is compared with traditional 3D static and fed-batch culture methodologies. The novel system performed better, producing higher alkaline phosphatase activity, and more homogeneous, dense and functional bone constructs. Additionally, cell constructs produced by the in-house-designed system were richer in mature osteoblast-like and mineralizing osteocyte-like cells. In conclusion, this study reports the development of a novel bioprocess able to produce hDPSC-based bone-like constructs, providing new insights into hDPSCs’ therapeutic potential and a system able to be transferred from the laboratory bench into medical facilities. Full article

Background: As a broad-spectrum fluoroquinolone, enrofloxacin (ENR) is commonly employed to manage bacterial infections in aquatic species. Nevertheless, there have been no documented pharmacokinetic and residue studies conducted on Dybowski’s frog (Rana dybowskii). Therefore, the objective of our study was to characterize the pharmacokinetics (PK) of ENR and its metabolite ciprofloxacin (CIP) in R. dybowskii, establish withdrawal times, and evaluate the physiological effects associated with ENR administration. Methods: Adult Rana dybowskii (120 individuals; 60 males and 60 females) were sex-separated and acclimated in four tanks. Prior to dosing, three males and three females were randomly selected as untreated controls (without ENR administration). Following the oral gavage of ENR (10 mg/kg), blood, liver, and kidney tissues were collected at 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 12, 24, 36, 48, and 72 h (n = 6) for pharmacokinetic analysis. Muscle and oviduct tissues were additionally sampled at 1, 3, 7, 15, and 30 days post-dose (n = 6) for ENR content determination. Serum/tissue ENR concentrations were measured via Liquid Chromatography–Tandem Mass Spectrometry (LC-MS/MS) and analyzed using a non-compartmental model (WinNonLin 6.1 software) to calculate PK parameters including peak time (T_max), peak concentration (C_max), and area under the curve (AUC_0−t). In studying the physiology effects of ENR administration, biochemical enzyme activities and gene expressions in the liver and intestine were assessed post-ENR administration. Results: ENR demonstrated rapid absorption and extensive distribution in R. dybowskii. The withdrawal periods were determined to be over 33 days for females and 34 days for males in R. dybowskii. Following ENR administration, there was an increase in immune enzymes (AKP (alkaline phosphatase) and ACP (acid phosphatase)) as well as glycolytic enzymes (HK (hexokinase), PK (pyruvate kinase), PFK (phosphofructokinase)). Antioxidant enzyme levels, specifically SOD (superoxide dismutase) and CAT (catalase), peaked at 1.5 h post-ENR administration but subsequently declined by the 8 h mark. Additionally, following ENR treatment, IGF1, PI3K, and Akt exhibited up-regulation, whereas Keap1 and GYS1 showed down-regulation. Conclusions: The administration of ENR at a dosage of 10 mg/kg significantly enhances the activities of AKP and ACP, promotes glycolysis, and activates the Keap1/Nrf2 and PI3K-Akt signaling pathways in R. dybowskii. These findings establish a foundation for the rational application of ENR and the determination of withdrawal times in frog aquaculture. Full article

Recent advancements in three-dimensional (3D) printing technology, particularly digital light processing (DLP) 3D printing, have enabled the customization of bone substitutes with specific shapes that match bone defect sizes and geometries. Magnesium calcium phosphate (MCP) has gained considerable attention due to its strong mechanical properties, degradability, and ability to promote bone regeneration. In this study, we prepared MCP samples with five different molar ratios via DLP 3D printing. We analyzed the physicochemical properties of these five groups, including phase compositions and microstructures, which were examined using X-ray diffraction and scanning electron microscopy, respectively. Additionally, we assessed the effects of MCP on material density and shrinkage. Biaxial flexural strength and degradation rate were evaluated; biological properties were examined through WST-8 analysis and alkaline phosphatase activity assays. Among the tested samples, MCP1/1 exhibited the highest strength. A higher proportion of magnesium phosphate in MCP corresponded to an increased degradation rate. Cell response observations in the WST-8 assay indicated that cell proliferation was better in the MCP1/1 group than in the other groups on days 4 and 7 of culturing. Alkaline phosphatase activity assays demonstrated that MCP1/1 exhibited higher activity than calcium phosphate. Our findings suggest that MCP1/1 can be used effectively in bone-tissue-engineering applications. Full article

Hypophosphatasia (HPP) is a rare inborn error of metabolism caused by pathogenic variants in ALPL, coding for tissue non-specific alkaline phosphatase. HPP patients suffer from impaired bone mineralization, and in severe cases from vitamin B₆-responsive seizures. To study HPP, we generated alpl^-/- zebrafish using CRISPR/Cas9 gene-editing technology. At 5 days post fertilization (dpf), no alpl mRNA and 89% lower total alkaline phosphatase activity was detected in alpl^-/- compared to alpl^+/+ embryos. The survival of alpl^-/- zebrafish was strongly decreased. Alizarin red staining showed decreased bone mineralization in alpl^-/- embryos. B₆ vitamer analysis revealed depletion of pyridoxal and its degradation product 4-pyridoxic acid in alpl^-/- embryos. Accumulation of d3-pyridoxal 5′-phosphate (d3-PLP) and reduced formation of d3-pyridoxal in alpl^-/- embryos incubated with d3-PLP confirmed Alpl involvement in vitamin B₆ metabolism. Locomotion analysis showed pyridoxine treatment-responsive spontaneous seizures in alpl^-/- embryos. Metabolic profiling of alpl^-/- larvae using direct-infusion high-resolution mass spectrometry showed abnormalities in polyamine and neurotransmitter metabolism, suggesting dysfunction of vitamin B₆-dependent enzymes. Accumulation of N-methylethanolaminium phosphate indicated abnormalities in phosphoethanolamine metabolism. Taken together, we generated the first zebrafish model of HPP that shows multiple features of human disease and which is suitable for the study of the pathophysiology of HPP and for the testing of novel treatments. Full article

Precisely localizing the spatial distribution of proteins within various brain cell types and subcellular compartments, such as the synapses, is essential for generating and testing hypotheses to elucidate their roles in brain function. While the fms-like tyrosine kinase-3 (Flt3) has been extensively studied in the context of blood cell development and leukemia pathogenesis, its role in the brain remains poorly understood. Previous efforts to address this issue were hindered by the low expression levels of Flt3 and the limited sensitivity of the standard immunolabeling method, which were insufficient to reliably detect Flt3 protein in brain tissue. In this study, we systematically characterized Flt3 protein localization during brain development using a highly sensitive immunolabeling method based on alkaline phosphatase (AP) polymer biochemistry. This approach revealed a previously unrecognized neuron-selective Flt3 expression pattern in both mouse and human cerebella, with a developmental increase in total protein levels accompanied by a shift from a cytosolic to a dendritic subcellular distribution. Combining AP-polymer-based immunohistochemistry (AP-IHC) for Flt3 with conventional immunostaining of cell type marker proteins revealed parvalbumin- and calbindin-positive Purkinje cells to be the main cell type expressing Flt3 in the cerebellum. To validate the versatility of the AP-IHC method for detecting low-abundance neuronal proteins, we demonstrated robust labeling of Kir2.1, a potassium channel protein, in brain tissue sections from mouse, pig, and human samples. We further applied the AP-IHC method to human stem cell-derived neurons, effectively visualizing the postsynaptic density scaffold protein PSD95 within synapses. To our knowledge, this is the first study to employ an AP-IHC method combined with other standard immunofluorescent staining to co-detect weakly expressed neuronal proteins and other cellular markers in brain tissue and cultured neurons. Additionally, our findings uncover a previously unrecognized neuron-specific pattern of Flt3 expression in the cerebellum, laying the foundation for future mechanistic studies on its role in normal brain development and neurological disorders. Full article

Background: The use of ex vivo assays associated with biomaterials may allow the short-term visualization of a specific cell type response inserted in a local microenvironment. Blood is the first component to come into contact with biomaterials, providing blood clot formation, being substantial in new tissue formation. Thus, this research investigated the physiological blood clot (PhC) patterns formed in 3D scaffolds (SCAs), based on chitosan and 20% beta-tricalcium phosphate and its effect on osteogenesis. Initially, SCA were inserted for 16 h in rats calvaria defects, and, after that, osteoblasts cells (OSB; UMR-106 lineage) were seeded on the substrate formed. The groups tested were SCA + OSB and SCA + PhC + OSB. Cell viability was checked by MTT and mineralized matrix formation in OSB using alizarin red (ARS). The alkaline phosphatase (ALP) and bone sialoprotein (BSP) expression in OSB was investigated by indirect immunofluorescence (IF). The OSB and PhC morphology was verified by scanning electron microscopy (SEM). Results: The SCA + PhC + OSB group showed greater cell viability (p = 0.0169). After 10 days, there was more mineralized matrix deposition (p = 0.0365) and high ALP immunostaining (p = 0.0021) in the SCA + OSB group. In contrast, BSP was more expressed in OSB seeded on SCA with PhC (p = 0.0033). Conclusions: These findings show the feasibility of using PhC in ex vivo assays. Additionally, its inclusion in the experiments resulted in a change in OSB behavior when compared to in vitro assays. This “closer to nature” environment can completely change the scenario of a study. Full article

Polycyclic aromatic hydrocarbons (PAHs) are known to have toxic effects on fish. In this study, we examined the effects of benz[a]anthracene (BaA), a type of PAH, on fish liver metabolism. Nibbler fish (Girella punctata) were intraperitoneally injected with BaA (10 ng/g body weight) four times over a 10-day period. BaA significantly decreased known bone metabolism-related plasma factors such as calcium and inorganic phosphorus. Moreover, significant reductions were observed in the plasma levels of known liver metabolism-related factors, including ferrous ions, total bile acids, total bilirubin, free bilirubin, aspartate aminotransferase, and alkaline phosphatase. Interestingly, mono-hydroxylated metabolites of BaA, such as 3 hydroxylbenz[a]anthracene (3-OHBaA), were detected in the bile of BaA-injected nibbler fish. This hydroxylated form of BaA was found in its free form, rather than conjugated with glucuronic acid or sulfuric acid. Due to the lack of whole-genome sequence data for the nibbler fish, two nibbler fish-specific apoptosis-related factors (TNF receptor superfamily member 1A: tnfrsf1a and TNF superfamily member 10: tnfsf10) were isolated by De novo RNA sequencing. In a liver tissue culture, 3-OHBaA (10⁻⁶ M) significantly upregulated the expression of tnfrsf1a and tnfsf10 in the liver. These results provide the first evidence that 3-OHBaA metabolites exhibit toxic effects on the liver in teleost. Full article

Background: With the global increase in metabolic disorders, identifying effective dietary strategies is crucial for enhancing health outcomes. While various health advantages of alkaline reduced water (ARW) have been documented, its specific impacts on glucose and lipid metabolism in both healthy and diabetic conditions are still not well understood. Methods: This study investigates how ARW affects carbohydrate and lipid metabolism in male Wistar rats, which were induced to develop glucose metabolism disorders through subcutaneous injections of nicotinamide and streptozotocin (STZ). The rats were allocated into four groups: one group received distilled water, another ARW, with similar arrangements for both non-diabetic and diabetic rats. Throughout the six-week experiment, the rats had unrestricted access to food and water. At the end of the study, blood and tissue samples were collected post-euthanasia for further analysis. Results: Non-diabetic rats consuming ARW experienced significant decreases in plasma glucose, triglycerides, cholesterol, insulin, leptin, and TBARS levels, along with reduced activities of hepatic hexokinase and intestinal sucrase. Meanwhile, there were increases in hepatic antioxidant enzyme activities, such as glutathione peroxidase and glucose-6-phosphate dehydrogenase, although glutathione levels decreased. In diabetic rats, ARW supplementation notably reduced plasma glucose and the glucose area under the curve, lowered hepatic glucose-6-phosphatase and intestinal disaccharidase activities, and raised hepatic GSH levels. Conclusions: These findings suggest that ARW supplementation significantly enhances glucose and lipid metabolism and boosts antioxidant activity in both non-diabetic and diabetic rats, indicating its potential as a therapeutic aid for managing metabolic disorders. Full article

Background: CI-RM6P has different binding sites with affinities for both M6P and IGF2, plays a role in the regulation of the TGF-β and IGF pathways that is important for controlling cell growth and differentiation. We hypothesize that previously synthesised derivative of M6P could be an alternative candidate for bone tissue regeneration in terms of higher binding affinity, stability in human serum, low cost and temporal delivery. Methods: CH₃-M6P is synthesised based on previously described protocol; mesenchymal origin of isolated DPSCs was assessed by flow cytometry and AR staining prior to alkaline phosphatase (ALP) activity test, qPCR to evaluate differentiation specific marker expression, immunofluoresence, and SEM/EDS to evaluate organic and inorganic matrix formation; and rat aortic ring model to evaluate angiogenic effect of molecule. Results: CH₃-M6P upregulated ALP activity, the expression of the ALP, Col1, RunX2, Mef2C, TGFβ1, TGFβ1R, TGFβ2, and Smad3 genes under osteogenic conditions. The results of immunofluorescence and SEM/EDS studies did not show enhancing effect on matrix formation. As we observed, the induction effect of CH₃-M6P on the expression of angiogenic genes such as SMAD3 and TGFβ1R, even under osteogenic conditions, within the scope of research, we checked the angiogenic effect of the molecule and compared it to VEGF, showing that the CH₃-M6P is really angiogenic. Conclusions: Our findings provide an important clue for the further exploration of the molecule, which can be necessary to enhance the capability of the commonly used osteomedium, possibly leading to the development of bone-forming drugs and has the potential to be a dual-functioning molecule for bone tissue engineering. Full article

Phosphatases are enzymes that catalyze the hydrolysis of phosphate esters. They play critical roles in diverse biological processes such as extracellular nucleotide homeostasis, transport of molecules across membranes, intracellular signaling pathways, or vertebrate mineralization. Among them, tissue-nonspecific alkaline phosphatase (TNAP) is today increasingly studied, due to its ubiquitous expression and its ability to dephosphorylate a very broad range of substrates and participate in several different biological functions. For instance, TNAP hydrolyzes inorganic pyrophosphate (PP_i) to allow skeletal and dental mineralization. Additionally, TNAP hydrolyzes pyridoxal phosphate to allow cellular pyridoxal uptake, and stimulate vitamin B6-dependent reactions. Furthermore, TNAP has been identified as a key enzyme in non-shivering adaptive thermogenesis, by dephosphorylating phosphocreatine in the mitochondrial creatine futile cycle. This latter recent discovery and others suggest that the list of substrates and functions of TNAP may be much longer than previously thought. In the present review, we sought to examine TNAP within the alkaline phosphatase (AP) superfamily, comparing its sequence, structure, and evolutionary trajectory. The AP superfamily, characterized by a conserved central folding motif of a mixed beta-sheet flanked by alpha-helices, includes six subfamilies: AP, arylsulfatases (ARS), ectonucleotide pyrophosphatases/phosphodiesterases (ENPP), phosphoglycerate mutases (PGM), phosphonoacetate hydrolases, and phosphopentomutases. Interestingly, TNAP and several ENPP family members appear to participate in the same metabolic pathways and functions. For instance, extra-skeletal mineralization in vertebrates is inhibited by ENPP1-mediated ATP hydrolysis into the mineralization inhibitor PP_i, which is hydrolyzed by TNAP expressed in the skeleton. Better understanding how TNAP and other AP family members differ structurally will be very useful to clarify their complementary functions. Structurally, TNAP shares the conserved catalytic core with other AP superfamily members but has unique features affecting substrate specificity and activity. The review also aims to highlight the importance of oligomerization in enzyme stability and function, and the role of conserved metal ion coordination, particularly magnesium, in APs. By exploring the structural and functional diversity within the AP superfamily, and discussing to which extent its members exert redundant, complementary, or specific functions, this review illuminates the evolutionary pressures shaping these enzymes and their broad physiological roles, offering insights into TNAP’s multifunctionality and its implications for health and disease. Full article

Background: Dental pulp (DP) is a connective tissue composed of various cell types, including fibroblasts, neurons, adipocytes, endothelial cells, and odontoblasts. It contains a rich supply of pluripotent stem cells, making it an important resource for cell-based regenerative medicine. However, current stem cell collection methods rely heavily on the enzymatic digestion of dissected DP tissue to isolate and propagate primary cells, which often results in low recovery rates and reduced cell survival, particularly from deciduous teeth. Methods: We developed a novel and efficient method to obtain a sufficient number of cells through a one-step cultivation process of isolated DP. After the brief digestion of DP with proteolytic enzymes, it was scratched onto a culture dish and cultured in a suitable medium. By day 2, the cells began to spread radially from DP, and by day 10, they reached a semi-confluent state. Cells harvested through trypsinization consistently yielded over 1 million cells, and after re-cultivation, the cells could be propagated for more than ten passages. Results: The proliferative and differentiation capacities of the cells after the 10th passage were comparable to those from the first passage. The cells expressed alkaline phosphatase as an undifferentiation marker. Similarly, they also maintained the constitutive expression of stem cell-specific markers and differentiation-related markers, even after the 10th passage. Conclusions: This method, termed “scratch-based isolation of primary cells from human dental pulps (SCIP)”, enables the efficient isolation of a large number of DP cells with minimal equipment and operator variability, while preserving cell integrity. Its simplicity, high success rate, and adaptability for patients with genetic diseases make it a valuable tool for regenerative medicine research and clinical applications. Full article

Background; Turmeric starch (TS) has gained significant attention due to its potential health benefits. Rich in resistant starch (RS) and higher in phosphorus, TS is anticipated to possess properties of high-phosphorus-type RS. Objectives; To understand the host physiology of TS, this study investigated the dose-dependent effects of TS on colonic fermentation in rats. Methods; Four experimental diets containing different levels of TS (5%, 10%, and 20% w/w) were formulated and fed to male Fischer 344 rats for two weeks and compared with rats fed a 0% TS diet (TS0). Results; Results showed that increasing the dose of TS resulted in reduced body weight gain, lower visceral tissue weight, and increased cecal mucin and IgA levels compared with the TS0 group. Further, fecal dry weight increased dose-dependently parallel to the starch excretion rate. Higher doses of TS resulted in increased short chain fatty acid (SCFA) production, specifically cecal acetate content, as well as in a dose-dependent decrease in the cecal pH level. However, this study did not observe a positive effect of TS on colonic alkaline phosphatase (ALP) activity, and the impact on small intestinal ALP activity remains unclear. Notably, beneficial bacteria such as the family Oscillospiraceae, genus Lachnospiraceae NK4A136 group, and Ruminococcus spp. were found to have been enriched in the TS-fed groups, further supporting the beneficial effects of TS on gut microbiota and SCFA production. Additionally, the genus Mucispirillum, which is known to possess beneficial and opportunistic pathogenic traits under immunocompromised states, was found in the TS-fed groups. Conclusions; According to these results, it is clear that TS served as a prebiotic substrate in rats, with a notable modulation of the microbial composition. Full article