TMAO Supplementation to High-Carbohydrate Diet Reprogrammed Hepatic Metabolism and Intestinal Microbiota to Improve Liver Health and Disease Resistance of Micropterus salmoides

TL;DR

Adding TMAO to a high-carb diet in largemouth bass improves liver health, metabolism, and disease resistance through changes in gut bacteria and liver function.

Contribution

TMAO supplementation is shown to reprogram hepatic metabolism and enhance intestinal microbiota for improved fish health.

Findings

TMAO reduced glycogen accumulation in the liver by altering gene expression related to glycogen metabolism.

TMAO increased synthesis of long-chain fatty acids and amino acids while decreasing stress-related cortisol levels.

TMAO enhanced disease resistance against Nocardia seriolae by boosting immune gene expression and reducing liver damage.

Abstract

This study aimed to evaluate the effects of trimethylamine oxide (TMAO) supplementation (0.5% and 1%) to a high-carbohydrate diet on the growth performance, liver health, hepatic metabolome, intestinal microbiota and disease resistance of largemouth bass (Micropterus salmoides). After an eight-week feeding trial with three replicates, fish fed with TMAO-supplemented diets showed growth-promoting potential with increased difference with a prolonged rearing period. Importantly, TMAO supplementation significantly improved liver structure and function, with reduced intrahepatic glycogen accumulation due to reprogrammed glycogen metabolism, including down-regulated gys2 and ugp2b but up-regulated pygl expression levels. Targeted liver metabolomics analysis indicated the enhanced synthesis of long-chain fatty acid and amino acid in the 1% TMAO group, accompanied by decreased cortisol, indicating the attenuation of the stress response. Furthermore, TMAO supplementation changed the structure of the intestinal microbiota and particularly the intestinal content of Romboutsia, an important probiotic that can effectively utilize different kinds of dietary carbohydrate, showed an increasing trend with the increased TMAO supplementation levels. Finally, after sampling, all remaining fish were challenged with Nocardia seriolae. TMAO supplementation significantly enhanced the immune clearance function of largemouth bass against invading N. seriolae, with alleviated granulomatous nodules within liver but enhanced hepatic expression levels of nlrp3, caspase1, il-1β and il-18. These results collectively underscore the finding that TMAO may promote intestinal Romboutsia growth and reprogram hepatic metabolism to improve liver health, giving TMAO potential as a feed additive for growth and health promotion in largemouth bass.

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1. Introduction

Carbohydrates are the main energy source for animals, and an appropriate dietary carbohydrate intake is essential for animal growth and health [1]. In aquaculture practice, the inclusion of carbohydrate in the diet has been shown to exhibit protein-saving effects in several fish species [2], as its better potential energy supply could reduce the need to use energy from dietary protein. Moreover, carbohydrate content is required in the preferred extruded feeds in order to meet the floating property requirements [3]. Thus, adequate inclusion of dietary carbohydrate is both requisite and economic in the fish feed industry. The carbohydrate tolerance level in different fish species varies depending on their feeding habits; normally, herbivorous fish tolerate a higher carbohydrate level than omnivorous fish and omnivorous fish tolerate a higher carbohydrate level than carnivorous fish [4]. However, even in herbivorous fish species, excessive intake of carbohydrates significantly inhibited fish growth performance by reducing feed intake and impairing the utilization of other dietary nutrients [5]. Moreover, fish tissue health and immunity are always diminished when they are fed with excess carbohydrate. For example, in blunt snout bream (Megalobrama amblycephala), excess dietary starch resulted in increased blood glucose concentration, hepatic insulin resistance, excess accumulated glycogen or lipids in hepatocytes, and impaired liver function [6]. In fact, the over-intake of carbohydrate results in the excess flow of glucose to the liver, which is mostly converted into lipids via intermediate metabolic processes, while a small amount is used to synthesize glycogen. In channel catfish, a high-carbohydrate diet (HCD) usually induces glycogen content and lipid accumulation in the liver [7], resulting in increased viscerosomatic index (VSI) and hepatosomatic index (HSI). However, unlike other fish species, our previous study identified high carbohydrate diet-induced glycogenic hepatopathy rather than fatty liver disease or hepatic steatosis via liver biopsy and high-throughput metabolomics in largemouth bass, a carnivorous fish species of great economic value [8].

Considering the significant influences of excess carbohydrate on liver health, dietary interventions with functional components for the prevention of metabolic diseases are important [9]. Herein, a feed additive named trimethylamine oxide (TMAO) was assessed for its role in combating glycogenic hepatopathy of largemouth bass (Micropterus salmoides). As a counteracting solute within the cytoplasmic matrix, TMAO exists in multiple organismal groups including bacteria, plants, aquatic animals, and humans, and is accumulated as a cytoprotectant in response to both endogenous and environmental stressors [10]. Moreover, TMAO has been proven to function as a protein stabilizer [11], as it restores the normal function of mutant or diseased proteins via facilitating the correct folding of unstructured proteins, repairing misfolded proteins, and inhibiting the formation of harmful higher-order protein oligomers [12]. TMAO content is especially high in marine animals, and serves as a depth-related osmolyte [13], and studies have shown that TMAO promotes adaptation to the deep sea by protecting cellular functions and potentially reducing protein turnover [14]. In aquaculture practice, TMAO has normally been used as a feed stimulant in fish diets [15]. Recently, multiple studies have exhibited the beneficial role of dietary TMAO inclusion in improving growth performance, nutrient absorption and metabolism, and in maintaining the gut microbiota homeostasis of Megalobrama amblycephala [16]. In addition to its role in growth and nutrient metabolism, TMAO supplementation has also been reported to alleviate fish disease. For example, dietary TMAO inclusion at 1% significantly decreased the presence of skin lesions in rainbow trout when farmed in seawater [17]. Moreover, TMAO has also been reported to suppress soy-enteritis in rainbow trout at moderate inclusion levels [18]. In vitro, TMAO accumulation has been shown to suppress the HSP70 response in cultured fish cells [19] and tissues [20,21]. Moreover, TMAO has been shown to promote growth by enhancing digestive enzyme activities and promoting protein folding in the muscle tissue of Penaeus vannamei [22], and to improve the survival rate of the swimming crab (Portunus trituberculatus) under low-salinity stress [23]. In largemouth bass aquaculture practice, liver health is not only challenged by diet, but also endangered by the Nocardia seriolae-caused nocardiosis and other viral diseases [24]. N. seriolae is an intracellular Gram-positive pathogen that is susceptible to infection in immunocompromised and surface-damaged fish, with high morbidity and mortality rates [25]. In this study, an eight-week feeding trial was conducted to evaluate the role of TMAO supplementation at 0.5% and 1% in combating glycogenic hepatopathy in largemouth bass by analyzing growth performance, liver histology, metabolomics and gut microbiota. Then, fish were challenged with N. seriolae to examine the effect of TMAO supplementation on fish immunity and disease resistance.

2. Materials and Methods

2.1. Ethics Statement

The animal study was reviewed and approved by Animal Experiment Committee of Huazhong Agricultural University (permit number HZAUFI-2016-007).

2.2. Experimental Animal and Diet Preparation

Juvenile largemouth bass were purchased from a fish fry hatchery in Yichang, Hubei Province. The fish were acclimated for two weeks at the aquaculture base of Huazhong Agricultural University, which is equipped with a water recirculation system featuring temperature control and extensive biofiltration. After a 2-week acclimation period, 180 fish with an initial average body weight of (48.00 ± 0.50) g were randomly distributed into nine 400 L experimental tanks connected to a recirculating water and oxygen supply system. Three tanks were randomly assigned to be the same group which were fed with the same diet, with 20 fish reared in each tank.

In the present study, a basal experimental diet with a high carbohydrate level was designed, similar to that in our previous study [8]. Previous studies have exhibited the beneficial role of dietary TMAO inclusion levels from 0.2% to 1% for invertebrates and fishes [16,17,18,19,20,21,22,23], and thus another two TMAO-supplemented diets were designed with TMAO supplementation to a basal diet at 0.5% and 1% in the present study. The composition and nutritional levels of the three experimental diets are presented in Table A1. For the preparation of experimental diets, all raw ingredients were ground, sieved, and weighed according to the formulated ratios, and then thoroughly mixed, pelleted, air-dried, and stored at −20 °C until use.

2.3. Experimental Design

All 180 fish, placed in nine tanks for three groups, were first fed with three experimental diets for the rearing experiment. The water flow rate of the system was maintained at 0.5 L/min, the water temperature at 26 ± 2 °C, and the pH at 7.6 ± 0.2, and the dissolved oxygen saturation in the outlet water was kept above 85%. The photoperiod followed the natural light cycle of May to August in Wuhan, China. Feeding was conducted daily at 09:00 am and 16:00 pm. Each feeding session followed a “little and often” approach to avoid leftover feed, continuing until the fish exhibited obvious satiety. The amount of feed provided was recorded daily. After 2 weeks, 5 weeks and 8 weeks, all fish were counted and group-weighed under moderate anesthesia (3-aminobenzoic acid ethyl ester, MS 222 (Shanghai Aladdin Biochemical Technology Co., Ltd., Shanghai, China); 100 mg/mL) for growth parameter calculation.

At the end of the 8-week feeding trial, total fish number and body weight in each tank were counted and measured after the fish were anesthetized using 3-aminobenzoic acid ethyl ester (MS-222; 100 mg/mL). Then, six fish from each group were randomly selected for sampling. Blood was collected from the caudal vein using a syringe, followed by cardiac perfusion with ice-cold PBS to eliminate the influence of blood on the immune responses of the liver, kidney, and gill tissues. The liver, gill, and other tissues were then dissected. A portion of the tissues was fixed in 4% paraformaldehyde fixative, while another portion was embedded in OCT compound (Tissue-Tek^®^, Sakura Finetek, Torrance, CA, USA, Cat. #4583). All remaining tissue samples were immediately frozen in liquid nitrogen and stored at −80 °C for further analysis.

After sampling at the end of the feeding trial, all the remaining fish were then challenged with N. seriolae according to our previous study [26]. In simple terms, largemouth bass in each group were anesthetized with MS222 and then intraperitoneally injected with N. seriolae (100 µL × 10^7^ CFU/mL) recovered from a single colony using a shaker incubator at 180 rpm at 28 °C. After bacterial infection, all fish were transferred back to each tank, with the same water conditions. Sampling was conducted on the 1st, 3rd and 7th days post challenge, using methods similar to the above.

2.4. Histopathological Sectioning, H&E Staining and PAS Staining

After being fixed in paraformaldehyde for over 24 h, the liver tissues were transferred into gradient alcohol for dehydration, xylene for transparency, and then paraffin for embedding. Finally, the embedded tissues were sectioned with HM325 paraffin microtome (Thermo Fisher Scientific Inc., Waltham, MA, USA) to obtain 5 mm sections.

Sections were firstly stained with hematoxylin and eosin (H&E) according to the producers’ directions ( Servicebio Technology Co., Ltd., Wuhan, China). After dehydration, staining and cover-slipping with glycerin gelatin, the H&E-stained sections were observed and photographed under a BX53 fluorescence microscope (Olympus Corporation, Tokyo, Japan) at 20× magnification to assess the morphology of the hepatocytes.

Similarly, sections were then stained with periodic acid–Schiff (PAS) according to the producers’ directions (G1280, Solarbio, Beijing, China). After deparaffinization and hydration, the tissue sections were treated with an oxidizing agent and rinsed. Subsequently, the sections were stained with Schiff’s reagent. Following counterstaining with hematoxylin, the sections were dehydrated, cleared, and mounted with neutral balsam.

2.5. Biochemistry Assay and Analysis of Enzyme Activities

TMAO content in serum samples was assayed using liquid chromatography with tandem mass spectrometry (LC/MS/MS) according to Kong et al. 2024 [27]. Specifically, chromatographic separation was carried out on an Agilent 1290 Infinity UHPLC system (Agilent Technologies Inc., Santa Clara, CA, USA), and mass spectrometric detection was achieved with a 5500 QTRAP mass spectrometer (AB Sciex, Framingham, MA, USA) operated in MRM mode. Briefly, 10 µL serum samples were added to 40 µL of acetonitrile containing d9-TMAO (50 ng/mL), and were vortexed and centrifuged 2 times (16,900× g, 5 min). Three microliters of supernatant was obtained for LC/MS/MS analysis.

The glycogen content in liver tissue was determined using a Glycogen Content Assay Kit (BC0345, Solarbio, Beijing, China). The glycogen content was calculated as follows: Glycogen (mg/g tissue) = (C_standard_ × V1) × (A3 − A1) ÷ (W × V1 ÷ V2) ÷ 1.11 = 0.450 × (A3 − A1) ÷ (A2 − A1) ÷ W (sample weight).

The alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity in the serum of largemouth bass was measured using commercial assay kits (BC1555&BC1565, Solarbio, Beijing, China), following the manufacturers’ instructions. One unit of ALT/AST activity was defined as the amount of enzyme that catalyzes the production of 1 μmol of pyruvate per hour per milliliter of serum.

2.6. RNA Extraction, cDNA Synthesis, and Quantitative Real-Time PCR

Total RNA was extracted from tissue samples using TRIzol^®^ Reagent (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). The purity and concentration of the RNA were assessed with a NanoPhotometer NP80 Touch ultra-micro spectrophotometer (Implen GmbH, Munich, Germany), with integrity verified by 1.0% agarose gel electrophoresis. First-strand cDNA, suitable for open reading frame cloning, was synthesized using a Yeasen reverse transcription kit (Yeasen, Shanghai, China). Quantitative real-time PCR (qRT-PCR) was performed using a Roche LightCycler^®^ 480 system (Roche Diagnostics, Jena, Germany). The 18S rRNA gene was used as the internal reference gene. Relative quantitative data were analyzed using the 2^−ΔΔCt^ method. All qRT-PCR data were analyzed with GraphPad Prism 6 software.

2.7. High-Throughput Targeted Metabolomics Analyses in Largemouth Bass Liver and Serum

Targeted metabolomic analyses of the liver of largemouth bass (n = 5 per group) were conducted to quantify global metabolites in HC and 1% TMAO groups. The analyses were carried out by Applied Protein Technology Co., Ltd. (Beijing, China). Tissue metabolites were profiled using an AB 6500+ QTRAP mass spectrometer (AB SCIEX, Framingham, MA, USA), while serum TMAO was absolutely quantified using an Agilent 1290 UHPLC system (Agilent, Santa Clara, CA, USA) coupled with a 5500 QTRAP mass spectrometer, both operating in MRM mode. Data were processed with MultiQuant software (version 3.0.2) for peak integration and quantification based on standard curves. Subsequent statistical analysis included univariate and multivariate tests, screening for differential metabolites, correlation analysis, and KEGG pathway enrichment.

2.8. 16S Sequencing

Total genomic DNA was extracted from microbial communities using the TIANGEN Genomic DNA Kit (TIANGEN, Beijing, China). The DNA quality was verified by 1% agarose gel electrophoresis, and concentration/purity were measured with a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, DE, USA). The V3–V4 region of the 16S rRNA gene was amplified with barcoded primers 338F and 806R. PCR products were purified and quantified, and sequencing was carried out using an Illumina PE300/PE250 platform (Illumina, San Diego, CA, USA).

Raw sequencing reads were quality-controlled and assembled. Operational Taxonomic Units (OTUs) were clustered at 97% similarity, and chimeras were removed. All samples were rarefied to an even sequencing depth. Taxonomy was assigned using the RDP classifier against the SILVA database (v138). Functional prediction was performed using PICRUSt2 (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States). Then, all analyses were conducted on the Majorbio Cloud Platform. Alpha diversity indices (Chao1, Shannon) were calculated using Mothur v1.30.1, and group differences were assessed with the Wilcoxon test. Beta diversity was analyzed via PCoA based on Bray–Curtis distances.

2.9. Statistical Analysis of Data

The following formulas were used for growth performance calculations:

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All data were statistically analyzed using GraphPad Prism 9 software. The data are presented as the mean ± SEM from at least three independent experiments. Differences among three groups were analyzed by one-way ANOVA to identify the effects of TMAO supplementation. The homogeneity of variance test was conducted to ensure that variance was homogeneous. Tukey’s test was utilized to compare individual means. A p-value of less than 0.05 was considered statistically significant (* p < 0.05, ** p < 0.01, *** p < 0.001).

3. Results

3.1. TMAO Supplementation to High-Carbohydrate Diets Did Not Significantly Affect Growth Performance in Juvenile Largemouth Bass

In order to systematically evaluate the growth performance among three groups, three growth parameters, including MFW, WGR and SGR, were evaluated from 0 to 2 weeks, 3–5 weeks and 6–8 weeks (Figure 1). Results showed that no significant differences were observed in MFW, WGR, or SGR among the three groups at the end of the three feeding periods (p > 0.05). Moreover, the FCR of largemouth bass in three groups at the end of the 8-week feeding trial also showed no significant difference, with results of 1.11 ± 0.02, 1.08 ± 0.03, and 1.09 ± 0.04 in the three groups. However, it is clear that with prolonged feeding duration, the differences in MFW, WGR, and SGR between the TMAO-supplemented groups and the HC group became more pronounced, indicating that TMAO supplementation may exhibit a significant growth-promoting role in largemouth bass with a longer rearing period.

3.2. TMAO Supplementation to High-Carbohydrate Diets Improved Liver Histological Structure and Function in Juvenile Largemouth Bass

As shown in Figure 2A, the serum TMAO content was significantly higher in the 1% TMAO group than that in the HC (p < 0.05), while no significant difference was found between the HC and 0.5% TMAO groups. Moreover, TMAO supplementation at both 0.5% and 1% TMAO significantly reduced the serum activities of ALT and AST (p < 0.05), while no significant difference was detected between two TMAO-supplemented groups.

Furthermore, the liver histological structure of largemouth bass fed with three experimental diets was evaluated via HE staining (Figure 2C). Irregular arrangement of hepatic plates, disruption and distortion of hepatic cords, blurred boundaries of hepatic lobules, and partial destruction of the lobular architecture were detected in the HC group (Figure 2C(a–c)). However, these pathological changes were markedly ameliorated by TMAO supplementation. Clear cord-formed cell plates could be detected with hepatocytes radially arranged around the central vein in both 0.5% TMAO and 1% TMAO groups (Figure 2C(d–i)). ALT and AST enzyme activities were also measured.

3.3. TMAO Supplementation to High-Carbohydrate Diet Reduced Excess Intrahepatic Glycogen via Reprogramming Glycogen Metabolism in Juvenile Largemouth Bass

PAS staining on the liver sections was conducted to detect the intrahepatic glycogen. As shown in Figure 3A, liver in the HC group displayed strong pink staining across the entire field, and hepatocytes were densely packed purplish-red granules, indicating excess glycogen accumulation in largemouth bass liver. However, glycogen in the liver of 0.5% TMAO and 1% TMAO groups was uniformly distributed within the hepatocyte cytoplasm, presenting as a fine, even light pink background. Furthermore, a glycogen assay kit was adopted to quantitatively measure the hepatic glycogen content. The results were glycogen contents in 0.5% TMAO and 1% TMAO of 150.86 ± 3.87 mg/g and 167.13 ± 2.20 mg/g, which were significantly lower than the 184.21 ± 0.92 mg/g in the HC group. Thus, TMAO supplementation at 0.5% and 1% markedly ameliorated the glycogen accumulation induced by a high-carbohydrate diet.

In order to illustrate the glycogen metabolism in three groups, the expression of genes involved in glycogen synthesis and catabolism was evaluated via RT-qPCR. As shown in Figure 3C, the expression of gys2 and ugp2b, responsible for glycogen synthesis, was significantly down-regulated in both TMAO-supplemented groups compared to the HC group. In contrast, the expression of pygl, the gene promoting glycogen breakdown, was significantly up-regulated. Moreover, the expression levels of g6pd, which was the key initiating enzyme of the pentose phosphate pathway, were significantly increased in 0.5% and 1% TMAO groups compared to the HC group, indicating that excess carbohydrate may be used to provide NADPH and pentose phosphate for cells.

3.4. TMAO Supplementation to High-Carbohydrate Diet Reprogrammed Hepatic Metabolism in Juvenile Largemouth Bass

High-throughput targeted metabolomics analyses with liver samples from HC and 1% TMAO groups were performed using the H650 platform to further investigate the regulatory mechanism of TMAO supplementation on the hepatic metabolism in largemouth bass. OPLS-DA score plots revealed a clear separation in metabolite profiles between the 1% TMAO group and the high-carbohydrate (HC) group (Figure 4A). Volcano plot analysis and bar charts indicated that 1% TMAO supplementation significantly increased the amounts of metabolites including linoleoyl ethanolamide, myristelaidic acid, and 4Z,7Z,10Z,13Z,16Z-docosapentaenoic acid, while 1% TMAO supplementation significantly decreased the amounts of metabolites including serine, cortisol, and 2,6-diaminopimelic acid (Figure 4B,C). Correlation heatmap analysis indicated the positive correlation between linoleoyl ethanolamide and myristelaidic acid, and the correlation between serine and 2,6-diaminopimelic acid (Figure 4D).

Further KEGG pathway enrichment analysis showed that the differential metabolites were predominantly enriched in the biosynthesis of amino acid pathways (Figure 4E). To assess the overall changes in metabolic pathways, a Differential Abundance (DA) score plot was generated, which indicated minimal overall perturbation in the global pathways.

3.5. TMAO Supplementation to High-Carbohydrate Diets Modulated the Microbiota Composition in Largemouth Bass Intestine

The gut microbiota composition of the three groups was assessed via 16S rRNA sequencing. The assembly operational taxonomic units (OTUs) can be classified into 27 phyla, 58 classes, 131 orders, 211 families, 372 genera, and 505 species. Then, community richness indices (ACE and Chao1 indices) and community diversity indices (Shannon and Simpson indices) in three groups were calculated to determine the alpha diversity, which measures species richness and evenness. The ACE, Chao1, Shannon, and Simpson indices showed no significant differences among HC, 0.5% TMAO, and 1% TMAO groups (Figure 5A), indicating that TMAO supplementation (0.5% and 1%) to the high-carbohydrate diet did not significantly change either the richness or the diversity of the gut bacterial community in largemouth bass (Figure 5B). Principal Coordinate Analysis (PCoA) and Non-metric Multidimensional Scaling (NMDS) were employed to assess the effects on the structural variation in the gut microbiota composition among three groups. The PCoA plot, based on the Bray–Curtis distance algorithm, clearly showed distinct and separate clusters in the coordinate space formed in the three groups (HC, 0.5% TMAO, and 1% TMAO). The HC group was completely separated from the two TMAO-treated groups along the PC1 axis, while a separation trend between the 0.5% TMAO and 1% TMAO groups was observed along the PC2 axis, suggesting that TMAO supplementation exhibited a dose-dependent effect on the microbial community structure. NMDS analysis based on the weighted unifrac distance algorithm also revealed a clear separation of the gut microbial communities among the different dietary treatment groups (stress = 0.026). In particular, the increased spatial separation between the 1% TMAO and HC groups compared to that between the 0.5% TMAO and HC groups further confirmed a dose-dependent response of TMAO on the core microbial community composition (Figure 5B). Collectively, these results suggest that TMAO supplementation caused a change in the centroid position of the gut microbiota in largemouth bass fed with a high-carbohydrate diet.

Further study indicated that TMAO supplementation changed the microbial abundance at both the phylum level and the genus level. At the phylum level, Firmicutes, Fusobacteriota, and Cyanobacteria were the dominant phyla across all three groups. The ratio of firmicutes to bacteroidetes showed an increasing trend with the increased TMAO supplementation level, which was significantly higher in the 1% TMAO group than in the HC group (p < 0.05). At the genus level, the relative abundances of the dominant genera were also altered by TMAO supplementation. The relative abundances of Staphylococcus, Weissella, Bacillus, Lactobacillus, Geomicrobium, Acinetobacter and Escherichia-Shigella showed a trend of decreasing with the increased TMAO level. The relative abundance of Cetobacterium was increased in the 0.5% TMAO group, but then decreased in the 1% TMAO group. However, the relative abundance of Turicibacter in the 1% TMAO group was significantly higher than that in the HC group (1.03% vs. 4.28%, p < 0.05). Furthermore, the relative abundance of Romboutsia, an important probiotic which has been reported to effectively utilize different kinds of dietary carbohydrate, exhibited a highly sensitive response to TMAO levels. Compared to that in the HC group (0.26%), the abundance of Romboutsia in the 0.5% and 1% TMAO groups increased to 1.38% and 5.28%, respectively. The 20.3-fold increase in the 1% TMAO group may that TMAO plays a key role in driving its colonization and proliferation (Figure 5C).

3.6. Effects of Supplementing High-Carbohydrate Diets with Different Levels of TMAO on the Liver Response of Largemouth Bass to N. seriolae Infection

The bacterial challenge experiment with N. seriolae injection was conducted in three groups of largemouth bass after the 8-week feeding trial. H&E staining identified increased inflammatory cell infiltration in liver since 1 dpi, which was exacerbated by a prolonged infection period. Moreover, small distinct granulomatous nodules were seen in the liver tissue of the HC group at 7 days post infection (dpi). The formation of these granulomatous nodules was alleviated in the two TMAO-supplemented groups (Figure 6A). Meanwhile, the expression of immune-related genes in the liver was detected by qRT-PCR. Figure 6B indicated that the expression of caspase1, il-1β and il-18 showed a trend to increase at 1 day post infection. At 1 dpi, the highest expression of caspase1 and il-18 at 1 dpi was detected in the 0.5% TMAO group, followed by the 1% TMAO group, and the lowest expression was detected in the HC group. Although the highest expression of il-1β at 1 dpi was detected in the HC group, its expression at 3 dpi and 7 dpi reached the highest level in the 0.5% TMAO group. Similarly, the highest expression of nlrp3 was detected in the 1% TMAO group at 1 dpi, and its expression at 7 dpi reached the highest level in the 0.5% TMAO group. Thus, at 7 dpi, the 0.5% TMAO group showed the highest expression level of all four detected genes, including nlrp3, caspase1, il-1β and il-18.

4. Discussion

TMAO has shown growth-promoting potential as a feed additive in several aquatic species, such as shrimp [22], crabs [28], and some teleost fish (e.g., tilapia [29] and Taimen [30]). However, its effects appear inconsistent across species, with studies in M. amblycephala [16] and rainbow trout [18] reporting no significant impact on growth. Consistent with the latter findings, our present study demonstrated that supplementing HC-based diets with 0.5% or 1% TMAO did not significantly affect the growth performance (MFW, WGR, SGR) of largemouth bass within the 8-week feeding period. In order to further illustrate the growth-promoting potential, the growth performance was evaluated every two or three weeks, showing the growth dynamics more accurately and capturing potential transient effects that might be missed with a single endpoint measurement [26]. Results showed that fish fed diets supplemented with TMAO exhibited a trend toward improved SGR and WGR with the prolonged rearing period, though the improvement did not reach statistical significance, indicating that TMAO supplementation may exhibit a significant growth-promoting role in largemouth bass with a longer rearing period. Thus, the growth benefits of TMAO may be time-dependent and show species-specific variations, requiring extended exposure and more fish species to manifest significant effects.

Our previous study has confirmed the hepatotoxic effects of high-carbohydrate diets in largemouth bass, as evidenced by the increased hepatocyte size, damaged cell membranes, and elevated serum ALT and AST levels [8]. In the present study, dietary TMAO supplementation markedly alleviated these pathological changes, maintaining hepatocyte integrity and reducing serum transaminase activities. Previous research on M. amblycephala also demonstrated that TMAO could protect liver function by reducing lipid accumulation and preserving hepatocyte ultrastructure integrity under high-carbohydrate stress [16]. In largemouth bass, our previous study confirmed that HC induced glycogenic hepatopathy rather than hepatic steatosis [8]. Thus, we analyzed the glycogen accumulation and metabolism in livers of fish fed with three diets. Both PAS staining and biochemistry analysis results showed that the glycogen content in bass liver was significantly decreased in TMAO-supplemented groups. This reduction was accompanied by a down-regulated expression of glycogen synthase (gys2) [31] and up-regulated glycogen phosphorylase liver isoform (pygl) [32] and its regulator, UDP-glucose pyrophosphorylase 2b (ugp2b) [33]. Thus, we saw enhanced glycogenolysis but suppressed glycogenesis in TMAO-treated fish, suggesting a metabolic shift favoring glycogen mobilization over storage. By promoting glycogen breakdown and limiting its synthesis, TMAO may effectively reduce the hepatic glycogen burden, thus protecting against liver dysfunction. Additionally, the expression of g6pd, a key pentose phosphate pathway enzyme [34], was significantly increased in the 0.5% and 1% TMAO groups, suggesting that TMAO may promote the pentose phosphate pathway to enhance antioxidant capacity and biosynthetic precursor supply. This metabolic shift could alleviate glycogenic hepatopathy by redirecting the glucose flux from glycogen storage to alternative pathways, and therefore maintaining hepatic homeostasis under high-carbohydrate conditions.

In order to illustrate the regulatory role of TMAO on the metabolism of bass liver, we conducted metabolomic analyses of liver in HC and 1% TMAO groups, revealing significant alterations in amino acid, lipid, and carbohydrate metabolism. TMAO supplementation notably increased the levels of linoleoyl ethanolamide and myristelaidic acid. Considering the regulatory role of linoleoyl ethanolamide in maintaining carbohydrate metabolism [35], the elevated linoleoyl ethanolamide level may also contribute to the stabilization of glucose homeostasis in bass under high-carbohydrate stress, thereby alleviating metabolic disturbances. Moreover, the level of 4Z,7Z,10Z,13Z,16Z-docosapentaenoic acid, an important omega-6 polyunsaturated fatty acid with potent total cholesterol-alleviating properties [36], was also significantly increased in the 1% TMAO group. Such metabolite enhancement may act synergistically to alleviate chronic inflammatory responses induced by a high-carbohydrate diet. KEGG enrichment analysis indicated the enriched biosynthesis of amino acid. The enhanced amino acid biosynthesis may reflect an adaptive response to metabolic stress, promoting efficient nutrient utilization and mitigating diet-induced dysregulation. Additionally, the cortisol level was decreased in the 1% TMAO group, indicating a potential attenuation of systemic stress response [37] which may further contribute to improved metabolic homeostasis and reduced inflammation; however, more physiological stress indicators should be measured in future studies to further confirm the results. This down-regulation of cortisol, in conjunction with the modulation of amino acid and lipid metabolism, suggests that TMAO supplementation at 1% could enhance adaptability to metabolic demands by balancing anabolic and catabolic processes. These findings collectively indicate that TMAO acts as a multifaceted osmolyte and metabolic modulator, enhancing hepatic adaptability under dietary stress.

Previous research on M. amblycephala showed that TMAO supplementation significantly changed the intestinal microbiota composition by decreasing the abundance levels of the genus Aeromonas and phyla Tenericutes and Bacteroidetes [16]. Here, the intestinal microbiota was also evaluated in three groups to further evaluate the role of TMAO supplementation. Figure 2A indicates increased TMAO content in the serum of bass fed with 1% TMAO, indicating increased circulating TMAO in largemouth bass, which is in accordance with a former study on TMAO content in the distal intestine showing an increasing trend with increasing TMAO inclusion rate, further alleviating soy-enteritis in rainbow trout [18]. Previous research also indicated that a higher firmicutes-to-bacteroidetes ratio in the gut microbiota is associated with the efficient absorption of energy from food [38]. In our study, the firmicutes-to-bacteroidetes ratio showed a trend of increasing with the increased TMAO supplementation level, indicating the potential for better utilization of dietary carbohydrate. Moreover, at the genus level, the proportions of Turicibacter and Romboutsia showed an increasing trend with the increased TMAO supplementation level, and 1% TMAO showed a significantly higher abundance of Turicibacter compared to that in the HC group (p < 0.05). The genomic analysis of the genus Romboutsia demonstrated the ability to grow effectively on L-fucose, glucose and sucrose [39], and Romboutsia was reported to prevent human diabetes [40] and ameliorate acute pancreatitis in rats [41]. A previous study on tilapia reported that the abundance of Romboutsia was positively correlated with dietary sugar levels, and Romboutsia-fermented feed regulated the glycometabolism of tilapia by producing SCFAs to stimulate insulin secretion and simultaneously reducing the carbohydrate content of feed to eventually decrease blood glucose [42]. Thus, the increased Romboutsia content in the intestine after TMAO supplementation may suggest its role in regulating the glycometabolism and carbohydrate catabolism to alleviate glucogenic hepatopathy induced by the high carbohydrate diet in largemouth bass. Moreover, the α-diversity (Ace, Chao1, Shannon and Simpson indices) showed no significant differences among the HC, 0.5% TMAO, and 1% TMAO groups. Although many studies have reported a correlation between higher α-diversity and good health [43], others have detected no [44] or even negative associations between α-diversity and the host condition (Davidson et al. 2021) [45]. Moreover, the β-diversity (PCoA and NMDS) revealed significant differences, and TMAO supplementation altered the structure of the gut microbiota in a dose-dependent manner (Figure 5B). Combining these results with the previously observed increase in Romboutsia, it is hypothesized that TMAO alters the overall structure of the intestinal microbiota by increasing the proportion of specific beneficial bacterial groups in the gut [42].

Considering the continuous exposure to pathogens [46] and other environmental stress [47], all the remaining largemouth bass from the three groups after sampling at the end of the 8-week feeding trial were injected with N. seriolae to detect the regulatory roles of TMAO in the largemouth bass’ defense against bacterial infection. The histological assay showed that the bass liver structure was significantly affected by infection, and this damage was exacerbated with the prolonged infection period; obvious granulomatous nodules in the liver tissue could be detected in the HC group at 7 dpi. However, such granulomatous nodules were rarely detected in the two TMAO-supplemented groups, indicating the protective role of TMAO in largemouth bass against N. seriolae infection. Early studies have indicated that N. seriolae infection mediates liver granulomatous chronic inflammation in largemouth bass through pyroptosis [48]. Thus, the expression levels of representative genes including nlrp3, caspase1, il-1β and il-18 [49] were detected in bass at 1, 3 and 7 days post infection. Similarly to our previous study [26] and other reports [48], their expression levels were significantly increased, especially at 7 days post infection. However, their expression at the same period was significantly affected by dietary composition. The 0.5% TMAO group showed the highest expression of all four genes at 7 dpi among the three groups, the highest expression of caspase1 and il-18 at 1 dpi, and the highest il-1β expression level at 3 dpi. All these results indicated that TMAO supplementation (especially at the 0.5% level) significantly enhanced the immune clearance function of largemouth bass against invading N. seriolae. This fish disease-alleviating role of TMAO is similar to that found in earlier studies on skin lesions and soy-enteritis in rainbow trout [17,18].

5. Conclusions

Dietary TMAO is an important feed additive with great growth- and health-promoting potential in largemouth bass. TMAO supplementation significantly alleviated high-carbohydrate-induced glycogenic hepatopathy to improve liver health via reprogramming hepatic glycogen metabolism, fatty acid and amino acid biosynthesis. The increased Romboutsia to maintain homeostasis of the intestinal microbiota may also contribute to the protective role of TMAO supplementation, which increased the immune clearance function of largemouth bass against invading N. seriolae. Future research for the fishing industry should systematically evaluate the appropriate dietary inclusion level of TMAO in different fish species and focus on the contribution of the intestinal microbiota to the beneficial role of TMAO.