The aim of this study was to investigate the potential effects of inulin on immune capacity, antioxidant capacity, and intestinal microflora in spotted sea bass fed a high-fat diet. A total of 360 juveniles were randomly assigned into six groups with three replicates per group and twenty fish per replicate. The six groups included a CK (Calvin Klein) group fed a normal fat diet, a group fed a high-fat diet (HF), and four groups fed a high-fat diet supplemented with 0.5% (G1), 1% (G2), 1.5% (G3), and 2% (G4) inulin, respectively. The experiment lasted for ten weeks. The results indicated that consumption of a high-fat diet resulted in oxidative stress injury, decreased immunity, abnormal intestinal histopathology, and an imbalance in intestinal flora in spotted sea bass compared to the CK group. However, compared to the HF group, supplementation with inulin significantly increased superoxide dismutase activity while decreasing malondialdehyde content. Notably, supplementation at 1.5% also led to significant increases in Complement 3 (C3) and Immunoglobulin M (IgM) levels while improving intestinal tissue morphology. Furthermore, phylum-level analysis revealed that Bacteroidetes, Proteobacteria and Firmicutes were the main bacterial groups found within the intestines of spotted sea bass. In terms of genus-level identification, Muribaculaceae, Citrobacte and Prevotellaceae_UCG-001 were identified as dominant bacterial groups. The abundances of Bacteroidetes and Muribaculaceae in the inulin group initially increased but then decreased with increasing supplementation amounts.
Research Article
Potential effects of inulin as a dietary supplement on immunity, antioxidant and intestinal health of spotted sea bass (Lateolabrax maculatus) fed a high-fat diet
https://doi.org/10.21203/rs.3.rs-4969844/v1
This work is licensed under a CC BY 4.0 License
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Inulin
Spotted sea bass
Immunity
Antioxidant
Intestine health
In intensive aquaculture, it is a common practice to increase the fat content of feed in order to conserve protein and reduce production costs, nitrogen emissions, and protect the aquaculture water environment. However, the physiological conditions of aquatic animals impose limitations on their ability to meet the demand for and effectively utilize fat. Excessive intake of fat often results in body fat accumulation, leading to metabolic disorders and disturbances in aquatic animals, ultimately impacting immunity, stress resistance, and the quality of aquatic products while causing economic losses (Gao et al. 2011; Jing et al. 2015). These factors significantly hinder the healthy and sustainable development of the aquaculture industry. Therefore, there is an urgent need to address this issue by ensuring the health status of aquatic animals while enhancing the economic benefits of aquaculture. Green environmental functional additives as a research hotspot may provide a regulatory solution.
Inulin is widely distributed among various plant species and consists of a fructose polymer linked by a β-2,1 glucoside bond. Its degree of polymerization (DP) typically ranges from 2 to 60 (Yuqing et al. 2023). Despite the limited presence of inulin in aquatic animal nutrition research, its extensive utilization in feed production has been well-documented. As a dietary supplement, it has been widely used in livestock and poultry breeding. Inulin itself can hardly be digested by the host, but it can be absorbed and utilized by beneficial microbes in its gut. Previous studies have shown that dietary inulin supplementation can significantly increase the secretion of immunoglobulin and inflammatory cytokines in goat ileum (Chunmei et al. 2023). Adding 1% inulin can significantly improve the antioxidant capacity of broilers (Elena et al. 2023). Furthermore, inulin can also alleviate the issue of bacterial imbalance caused by antibiotics (An et al. 2024), promote the growth of host intestinal probiotics such as Bifidobacterium and Turicibacter, and inhibit the proliferation of pathogenic bacteria Achromobacter (B 2007). Therefore, inulin, as a dietary supplement, has potential probiotic effects on many farmed animals.
The spotted sea bass (Lateolabrax maculatus) is a significant economic fish in China's inland and coastal regions. In recent years, due to the decline of fishery resources and increased demand for fishmeal, the price of fishmeal has remained high. Consequently, in the production process, it is commonly employed to augment fat content as a means of protein preservation. However, as mentioned earlier, the adverse effects associated with a high-fat diet on spotted sea bass culture cannot be disregarded; these include compromised immunity and antioxidant capacity. Therefore, this study aims to investigate the impact of inulin supplementation on immune response, antioxidant capacity, and intestinal health in spotted sea bass fed a high-fat diet by evaluating serum and tissue enzyme activity levels, conducting histopathological examination (HE), and analyzing 16S rRNA.
2.1 Experimental feed and production
According to the nutritional needs of the mandarin fish (Ai et al. 2004b), this experiment designed 6 groups of feed with fish meal and soybean meal as protein sources, and fish oil and soybean oil as fat sources. Among them, 3% fish oil and 2% soybean oil are the healthy group, labeled as CK;6% fish oil and 4% soybean oil are the positive control group, labeled as HF༛In addition, 5, 10, 15, and 20g/kg of inulin were added to the HF group separately, and formulated with microcrystalline cellulose, labeled as G1, G2, G3, and G4 (Table 1).
| Ingredients | Group | |||||
|---|---|---|---|---|---|---|
| CK | HF | G1 | G2 | G3 | G4 | |
| Fish meal | 48 | 48 | 48 | 48 | 48 | 48 |
| Soybean | 23 | 23 | 23 | 23 | 23 | 23 |
| Flour | 10 | 10 | 10 | 10 | 10 | 10 |
| Fish oil | 3 | 6 | 6 | 6 | 6 | 6 |
| Soybean oil | 2 | 4 | 4 | 4 | 4 | 4 |
| Yeast powder | 2 | 2 | 2 | 2 | 2 | 2 |
| Choline | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 |
| Ca(H2PO4)2 | 1 | 1 | 1 | 1 | 1 | 1 |
| Lecithin | 1 | 1 | 1 | 1 | 1 | 1 |
| Microcrystalline cellulose | 8.2 | 3.2 | 2.7 | 2.2 | 1.7 | 1.2 |
| Antioxidant | 0.3 | 0.3 | 0.3 | 0.3 | 0.3 | 0.3 |
| Mineral premixa | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 |
| Vitamin premixb | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 |
| Inulin | 0 | 0 | 0.5 | 1 | 1.5 | 2 |
| Total | 100 | 100 | 100 | 100 | 100 | 100 |
| Proximate composition (% dry weight) | ||||||
| Crude protein | 45.97 | 45.8 | 46.24 | 45.38 | 46.02 | 45.76 |
| Crude lipid | 10.1 | 15.6 | 15.0 | 15.2 | 15.0 | 15.0 |
| a | ||||||
Mineral premix: MnSO4·4H2O 50 mg/kg, MgSO4·H2O 4000 mg/kg, KI 100 mg/kg, CoCl2(1%) 100 mg/kg, CuSO4·5H2O 20 mg/kg, FeSO4·H2O 260 mg/kg, ZnSO4·H2O 150 mg/kg, Na2SeO3(1%) 50 mg/kg.
b
Vitamin premix: thiamine 25 mg/kg, riboflavin 45 mg/kg, pyridoxine hydrochloride 20 mg/kg, Vitamin B12 0.1 mg/kg, Vitamin K3 10 mg/kg, inositol 800 mg/kg, pantothenic acid 60 mg/kg, nicotinic acid 200 mg/kg, folic acid 20 mg/kg, biotin 1.2 mg/kg, vitamin A acetate 32 mg/kg, Vitamin D3 5 mg/kg, α-tocopherol 120 mg/kg, ethoxyquin 150 mg/kg.
The fish meal and soybean meal were pulverized using a high-speed grinder (SF130, Shanghai Tian Fan Pharmaceutical Machine Factory) with an 80-mesh screen. The feed raw materials were then mixed following a step-by-step method. Subsequently, fish oil, soybean oil, and lecithin were added and thoroughly mixed before passing through a 60-mesh screen. Afterwards, 30% water was added and thoroughly mixed before passing through a 40-mesh screen. The mixture was further processed using the granulator (SZJYJ-025, Xiamen Yin Fa Machinery Equipment Co., LTD.) to produce pellet feed with a particle size of 2.5mm. The pellets were dried in an oven at 55℃ until the moisture content reached approximately 10%. Finally, they were stored in a refrigerator at -20℃.
2.2 Experimental fish and feeding trial
After acquiring the experimental fish, they are temporarily housed in a tank (1200 L/barrel) for a duration of 2 weeks. During this temporary feeding period, the basic feed is provided twice daily, with timely suction and water changes. Daily water changes are gradually conducted to transition from saltwater to freshwater. Oxygen levels are maintained throughout the day, while maintaining a water temperature of 27℃ and dissolved oxygen concentration above 5.0 mg/L. The pH level is approximately 7.8 and ammonia nitrogen concentration remains below 0.3 mg/L during this period. Direct sunlight exposure is avoided during daylight hours, with no light provided at night. Following two weeks of temporary rearing, the selected fish intended for grouping undergo a fasting period of 24 hours before being divided into six groups consisting of three replicates per group and twenty individuals per replicate (totaling 360 healthy and vigorous spotted sea bass with relatively consistent specifications weighing around 2.35 ± 0.04 g). A total of eighteen test tanks (60 liters/tank) were utilized for a seventy-day culture experiment. The fish are fed twice daily at specific times (08:30 and 17:30), recording food intake per tank while siphoning bottom drains after each feeding session concludes. Water changes equivalent to forty percent of the aquaculture volume are performed thirty minutes after ceasing feeding to remove feces and other dissolved waste.
2.3 Sample collection
After a 24-hour fasting period, the fish were anesthetized with 150 mg/L eugenol (Holloway et al. 2004) at 9 am on the following day. Subsequently, blood samples were collected via tail vein puncture and stored in a refrigerator at 4℃ for approximately 18 hours. Following serum separation by centrifugation at 4℃ and 3500 r/min for 10 minutes, the resulting serum was stored in a -80℃ freezer for subsequent analysis of relevant serum enzyme activity indexes. Thereafter, gut and head kidney tissues from the aforementioned spotted sea bass with drawn blood were carefully excised using sterilized scissors and tweezers before being immediately preserved in liquid nitrogen. Upon completion of overall sampling procedures, the liquid nitrogen tank was transported into the laboratory where it was transferred to a -80 ℃ refrigerator for storage.
2.4 Detection and analysis of indices
2.4.1 Immunity parameters analysis
The immune indices measured in this experiment encompassed acidic phosphatase (ACP), alkaline phosphatase (AKP), lysozyme (LZM), C3, and IgM in both serum and head kidney. Additionally, intestinal ACP, AKP and LZM were assessed. The experimental kits utilized were procured from Nanjing Jiancheng Bioengineering Institute and employed following rigorous adherence to the provided instructions.
2.4.2 Antioxidant parameters analysis
Antioxidant indices including total antioxidant capacity (T-AOC), superoxide dismutase (SOD) activity, peroxidase (CAT) activity, reduced glutathione (GSH) content, and malondialdehyde (MDA) content in serum, head kidney, and gut were measured. The kits used in the experiment were purchased from Nanjing Jiancheng Bioengineering Institute and tested in strict accordance with the instructions.
2.4.3 Intestinal morphological observation
The intestinal tissue samples were fixed in 4% paraformaldehyde for 48 hours prior to section preparation. Subsequently, the tissue samples were flattened using a scalpel and rinsed with 75% ethanol. They were then sequentially soaked in increasing concentrations of ethanol (75%, 85%, and 90%) for 2 hours each, followed by dehydration in 100% ethanol for 1.5 hours. After dehydration, the tissues underwent transparentization using xylene treatment. The transparentized tissue blocks were embedded in molten paraffin wax and kept warm in a melting box until complete immersion of the wax block was achieved. Once the paraffin had solidified, the wax blocks were affixed onto a microtome and cut into thin slices which were subsequently ironed in hot water before being mounted on slides. Following drying in an incubator, dewaxing was performed by soaking the sections successively in xylene and ethanol solutions, followed by staining with hematoxylin and eosin dyes to visualize histological features. Finally, optical microscopy (Eclipse E100, Japan) coupled with WT-1000GM imaging system was employed to observe and capture images of the tissue sections while FIJI software was utilized for measuring villi height, width, and muscular thickness. Evaluation of intestinal histopathological changes was based on assessment of villi alterations.
2.4.4 Intestinal microbiome analysis
The total DNA of the intestinal flora was extracted using a DNA extraction kit (Beijing Tiangen Biochemical Technology Co., LTD). The quality of the total DNA was assessed by 1% agarose gel electrophoresis, and the concentration was determined using a Nano Drop 2000 ultraviolet-visible spectrophotometer (Thermo Scientific, USA). The high variable region V3-V4 of the 16S rRNA gene was amplified using forward primer 343F (5'-TACGGRAGGCAGCAG-3') and reverse primer 798R (5'-AGGGTATCTAATCCT-3'). PCR products were detected by 1% agarose gel electrophoresis and purified with the Omega DNA Purification Kit (Omega Inc., USA). After purification, PCR product quality was evaluated using Qsep-400 method. Sequencing was performed on an Illumina NovaSeq6000 High-throughput Sequencer (San Diego, CA USA) after passing quality control. Trimmomatic ver0.33 software sequentially filtered raw data from each group, while cutadapt 1.9.1 software identified and removed primer sequences. DADA2 in QIIME2(2020.11) denoised and removed chimeras to obtain final valid data. USEARCH10.0 software assigned eligible sequences with a similarity threshold greater than 97% to taxa. All OTU species information was classified and labeled based on naive Bayes classifier in QIIME2(2020.11) using SILVA database with a confidence threshold set at 70%. Finally, Alpha diversity analysis of each sample was conducted in QIIME2 (2020.11).
2.5 Data statistics analysis
The experimental data were expressed as Mean ± SD, and the difference between the HF group and CK group was analyzed using an independent sample T-test. One-way ANOVA was employed to compare the inulin supplementation group with both the HF and CK groups. Duncan's multiple comparison method was utilized to analyze significant differences between groups, and statistical significance testing was conducted with a threshold of (P < 0.05). All statistical analyses were performed using SPSS Ver27 software.
3.1 Immunity parameters analysis
The immune parameters related indicators are presented in Fig. 1, Fig. 2, and Fig. 3. Regarding the serum immune indicators, the IgM activity was significantly lower in both the HF group and the group with inulin addition compared to the CK group (P < 0.05). However, there were no significant differences observed in C3, LZM, AKP, and ACP activities between the HF group and CK group. The C3 activity was significantly higher in G2 and G3 groups compared to the HF group (P < 0.05), while ACP activity was significantly higher in G1 and G4 groups than that of the HF group (P < 0.05). The lysozyme content was significantly lower in G1, G2, and G3 groups compared to the HF group (P < 0.05). In comparison to the HF group, IgM and C3 activities were significantly higher in the G2 group (P < 0.05), whereas C3 and LZM activities were significantly higher in the G3 group (P < 0.05); however, no significant differences were observed among groups for ACP or AKP activities.
The results of immune indicators in the Head Kidney demonstrated a significant decrease (P < 0.05) in the activities of IgM, C3, AKP, and LZM within the HF group. However, upon addition of inulin to a high-fat diet, there was a notable increase (P < 0.05) observed in the activities of IgM, LZM, and C3 within the G3 group; an increase (P < 0.05) was also observed in AKP activity within the G4 group and LZM content within the G1 group when compared to the HF group. Furthermore, when comparing with CK group, there was a significant elevation (P < 0.05) noted specifically in LZM content within the G1 group. Notably, no significant difference was found among all six groups regarding ACP activity.
The results of intestinal immune indexes showed that there was no significant difference in the activity of LZM, AKP and ACP in CK group compared with HF group. Compared to the CK and HF groups, there were no significant differences observed in the activity of AKP and ACP within the inulin supplementation groups. However, it was found that the LZM content in G1 group was significantly lower than that in the CK group (P < 0.05), but not significantly different from that in the HF group. Nevertheless, an overall trend of initial increase followed by a subsequent decrease was observed.
3.2 Antioxidant parameters analysis
The antioxidant parameters related indicators are presented in Fig. 4, Fig. 5, and Fig. 6. The serum antioxidant indexes revealed that the HF group exhibited a significant increase in MDA activity (P < 0.05) and a significant decrease in GSH activity (P < 0.05) compared to the CK group, while no significant differences were observed for SOD, CAT, and T-AOC activities. Notably, only the G2 group showed a significantly increased activity in the GSH index (P < 0.05) without any notable variations observed among groups regarding SOD, MDA, CAT, and T-AOC activities. In comparison to the HF group, supplementation with inulin led to a significant increase in SOD activity (P < 0.05), displaying an initial increasing trend followed by a subsequent decline; moreover, it resulted in a significant decrease in MDA content (P < 0.05), exhibiting an initial decreasing trend followed by an eventual increase. Additionally, the G2 group displayed a significantly increased GSH activity (P < 0.05). No noteworthy differences were observed among all groups concerning CAT and T-AOC activities.
The results of the Head Kidney antioxidant indexes revealed a significantly higher MDA content in the HF group compared to the CK group (P < 0.05), while no significant differences were observed among groups in terms of SOD, T-AOC, GAT, and GSH activity. Compared to the CK and HF groups, there were no significant differences in the activities of SOD, MDA, T-AOC, and GSH in the inulin group. However, a significant increase was observed in CAT activity in the G1 group (P < 0.05).
The results of the intestinal index showed that compared to the CK group, the MDA content in the HF group was significantly increased (P < 0.05), while T-AOC, GSH, and CAT activity were significantly decreased (P < 0.05). However, there was no significant difference in SOD activity. In comparison to the HF group, the activities of T-AOC, GSH, and SOD were significantly increased in the G4 group (P < 0.05), whereas CAT activities were significantly increased in both G1 and G3 groups (P < 0.05). Additionally, the content of MDA was significantly decreased in the inulin group (P < 0.05). Furthermore, when compared to the CK group, there were significant increases observed in GSH, T-AOC and SOD activities within the G4 group (P < 0.05). Interestingly though after a significant increase in MDA content within the G1 group due to supplementation with inulin (P < 0.05), no significant decrease was observed within groups G2-G4.
3.3 Intestinal morphological observation
The observation results of intestinal tissue morphology are shown in Fig. 7, Fig. 8. The results demonstrated that the intestinal morphology of the CK group appeared normal, with intact and orderly epithelial cells, an intact lamina propria, and no observable lesions. In the HF group, there was shedding of the intestinal epithelium, lamina propria, and muscle layer along with perforation of the muscle layer. However, upon addition of inulin, improvements were observed in intestinal morphology. Although not statistically significant compared to the HF group, there was a noticeable increasing trend in muscle layer height within the inulin supplementation group. Furthermore, villus height significantly increased (P < 0.05) when comparing it to the CK group. There were no significant changes observed in villi or muscle thickness. Additionally, a small number of epithelial cells shed within the inulin supplementation group; however, their degree of lesion was significantly lower than that seen in the HF group.
3.4 Intestinal microbiota analysis
3.4.1 Microbial species analysis
At the phylum level, Bacteroidetes, Proteobacteria, and Firmicutes were identified as the dominant bacteria in the CK group. The composition of dominant flora in other groups exhibited similarities to that of the CK group. In comparison with the CK and HF groups, there was no significant difference observed in the proportion of Bacteroidetes within the inulin group; however, a trend of initial increase followed by decrease was noted with increasing dosage. The proportion of Proteobacteria was significantly lower in G2 and G3 groups compared to that in the CK group (P < 0.05), while no significant difference was found between G2 and HF group. At the genus level, Muribaculaceae, Citrobacter, and Prevotellace_ucg-001 were identified as dominant bacterial taxa. Among them, there was an observed trend of initial increase followed by decrease for Muribaculaceae flora within the inulin addition group.
Table 2. Proportion of intestinal microbes at phylum level (%).
|
Group |
Phylum level relative abundance (%) |
||
|
Bacteroidota |
Proteobacteria |
Firmicutes |
|
|
CK |
40.7±4.16ab |
35.15±2.82b |
19.39±7.17 |
|
HF |
42.4±7.97ab |
20.23±9.15a |
32.81±11.95 |
|
G1 |
34.19±1.55a |
37.76±5.32b |
23.56±4.98 |
|
G2 |
42.42±4.53ab |
22.96±5.91a |
25.24±11.09 |
|
G3 |
44.15±6.34b |
20.65±5.24a |
25.41±5.6 |
|
G4 |
34.15±1.78a |
29.93±8.43ab |
25.62±10.47 |
Note: The values of different upper indexes in the same column were significantly different (P < 0.05). Based on three repeated averages.
Table 3. Proportion of intestinal microbes at genus level (%).
Note: The values of different upper indexes in the same column were significantly different (P < 0.05). Based on three repeated averages.
3.4.2 Alpha diversity analysis
The PD_whole_tree in the HF group exhibited a significant reduction compared to the CK group (Fig. 10). In contrast, the ace, chao1, observed_species, and PD_whole_tree were significantly increased in both G2 and G4 groups when compared to the HF group. Additionally, there was a significant decrease in Goods_coverage within the G3 group. Notably, no significant differences were observed among all groups regarding Shannon and Simpson indices.
The proteins AKP, ACP and LZM are expressed in the serum, intestine, and head kidney as components of the non-specific immune response. These proteins play a crucial role in both vertebrate and non-vertebrate immune defense systems (Reyes-Becerril et al. 2008; Yin et al. 2014), serving as important indicators of immune function (Zhao et al. 2023). The complement system is an effector system widely involved in immune regulation and anti-microbial defense of the body, and has important significance for fish immunity. Among them, C3 can clear pathogenic microorganisms and activate inflammatory cells (Gong et al. 2021). In the context of specific immunity, IgM possesses the highest molecular weight and serves as a crucial immunoglobulin in gnathoid vertebrates. It represents the initial immunoglobulin synthesized during the evolutionary progression of gnathoid vertebrates (Boes 2000), playing a pivotal role in various effector functions such as complement fixation and regulation of bony fish cytotoxicity (Song et al. 2006). Previous studies have provided evidence that the addition of inulin to the diet can significantly enhance the functions of AKP, ACP, and LZM in Procambarus Cruzii's hepatopancreas (Fu et al. 2022). Additionally, it has been demonstrated to improve ACP and phenoloxidase (PO) activities in Litopenaeus vannamei's hepatopancreas, while also reducing the viral load and occurrence of white spot syndrome virus (WSSV) (Luna-González et al. 2012). In this study, the activity values of ACP, AKP, and LZM in the inulin group did not exhibit significant differences compared to those in the high-fat group. The observed variations may be attributed to fish species and breeding conditions. However, IgM content was higher with 1% and 1.5% inulin supplementation, while C3 content reached its peak with a 1.5% inulin supplementation. Similar to the findings of this study, dietary supplementation with inulin has been shown to enhance the immune response in White-leg shrimp (Luna-González et al. 2012; Zhou et al. 2020), as well as significantly improve serum LZM activity and levels of C3 and C4 in young carp (Cyprinus carpio) (Mousavi et al. 2016). Therefore, inulin can be utilized as an immunostimulant for enhancing immune function.
Reactive oxygen species (ROS) inflict oxidative injury by targeting intracellular proteins, nucleic acids, and lipids, thereby playing a pivotal role in cellular damage. However, the body's antioxidant system efficiently eliminates ROS generated by normal metabolic activities of cells under physiological conditions (Hoseinifar et al. 2021). As a byproduct of lipid peroxidation, MDA can induce structural damage to the cell membrane, impair its functionality, and ultimately result in cellular senescence or demise. Consequently, it serves as a reliable marker for quantifying oxidative stress levels (Xie et al. 2020). The activities of CAT, GSH, and T-AOC were significantly reduced in this study, while the levels of MDA in serum, intestine, and head kidney were significantly increased. These findings are consistent with previous studies on spotted sea bass (Zou et al. 2021), black carp (Jianhua et al. 2022) and Yellow River carp (Yuru et al. 2022). Previous investigations on piglets (Liang et al. 2022) and white shrimp (Zhou et al. 2020) have demonstrated that inulin can mitigate the adverse effects of exogenous stimulus-induced oxidative stress. In our experiment, we observed a significant increase in SOD activity and a significant decrease in MDA content upon supplementation with inulin. These results indicate that inulin possesses the ability to inhibit lipid peroxidation and enhance the antioxidant capacity of spotted sea bass.
The intestinal morphology and flora structure serve as crucial indicators of intestinal health. The primary function of intestinal villi is to facilitate nutrient absorption (R. et al. 2020; Xuexi et al. 2023). In order to preliminarily analyze the efficiency of nutrient absorption and the overall state of intestinal health, we examined the height and width of villi, along with the thickness of the intestinal muscle. In this study, it was observed that the intestinal villi structure of the CK group remained intact, with no detachment of the intestinal wall muscle layer. Conversely, in the HF group, significant damage to the intestinal villi was evident, characterized by reduced height and loss of propria integrity along with perforation of the muscle layer. Similar findings have been reported in grass carp (Liu et al. 2022) and zebrafish (Yajie et al. 2022), demonstrating a significant reduction in villi height, villi atrophy, villi adhesion, and villi shedding. In the inulin group, there was a substantial increase in villi height, reduced occurrence of membrana propria shedding phenomenon, and an overall improved structural integrity. These results suggest that inulin supplementation can enhance intestinal morphology under high-fat diet conditions and promote nutrient absorption.
The fish gut microbial community plays a pivotal role in maintaining intestinal health, facilitating intestinal development, defending against pathogen invasion, and optimizing nutrient digestion and absorption. In contrast to terrestrial animals, the gastrointestinal tract of fish is predominantly colonized by aerobic bacteria, facultative anaerobic bacteria, and obligate anaerobic bacteria (Llewellyn et al. 2014). At the Phylum level, Proteobacteria, Bacteroidetes, Actinobacteria, Firmicutes, and Fusobacterium are the predominant bacterial groups in the gastrointestinal tract of fish (Meng et al. 2019), such as carp (Ling et al. 2022), juvenile largemouth bass Micropterus salmoides (Guanglun et al. 2022) and European sea bass Dicentrarchus labrax (David et al. 2020). Among them, Proteobacteria is a characteristic microorganism associated with intestinal inflammation (Shin et al. 2015), typically dominating the intestines of marine carnivorous fish (Egerton et al. 2018);Bacteroides can serve as an indicator of animal intestinal health (J et al. 2009)༛Firmicutes and Actinomycetes play a crucial role in facilitating nutrient absorption and metabolism in fish (Leon 2014; O et al. 2002). At the genus level, Prevotella (C. et al. 2020), Muribaculaceae (Ziel Institute For Food Health et al. 2019), Actinomyces (Irene and Sandra 2015) and Fusobacterium (Brennan and Garrett 2019) are considered to be bacteria with prebiotic functions. In a high-fat diet, excessive fat can easily lead to a reduction in the abundance of Bacteroidetes in the intestine and an increase in the prevalence of Firmicutes. The predominance of Firmicutes over Bacteroidetes in the intestinal microbiota can enhance food calorie absorption efficiency, thereby contributing to obesity (Fischbach and Segre 2016; Turnbaugh et al. 2006). In this study, Bacteroides, Proteobacteria, Firmicutes, and Actinobacteria were identified as the predominant bacterial phyla in the intestinal microbiota. At the genus level, Muribaculaceae, Citrobacte, and Prevotellaceae_UCG-001 were found to be dominant. Compared to the CK group and HF group, the abundance of Bacteroidetes in the inulin-treated group initially increased and then decreased with increasing addition amounts. The highest abundance was observed when 1.5% of inulin was added. It has been reported that inulin is mainly decomposed by beneficial bacteria such as Lactobacillus and Bifidobacterium to produce volatile fatty acids (such as acetic acid, propionic acid and butyric acid) and organic acids (such as succinic acid and pyruvate) (M and R 2014), which, as an important bifidobacterium factor, can increase the abundance of bifidobacterium in the intestine (Rosa et al. 2019). In this study, we only detected a small amount of bifidobacterium in the CK group and inulin group, ranking 13th in the TOP15 genus. The proportion of bifidobacterium in the top 15 flora abundance is evidently low. However, when supplemented with inulin, it exhibits certain probiotic effects on spotted sea bass. Nevertheless, these effects do not correspond to the amount of inulin added and may even have adverse consequences. The earlier study, however, indicated that inulin was ineffective and potentially toxic for aquatic animals (Cerezuela et al. 2013). Therefore, the biological function of inulin should be assessed from various perspectives.
Previous research has indicated that dietary supplementation with inulin led to a significant increase in the alpha diversity of the intestines in mice (Zhang et al. 2017). The results of our study do not fully support this hypothesis. As shown in Fig. 9, it is evident that the addition of inulin resulted in elevated proportions of ace, chao1 and observed indices. Conversely, the coverage of Goods index decreased, while no significant differences were found in Simpson and Shannon indices between the groups. The composition of intestinal microbiota in fish is influenced by various complex factors (Liu et al. 2024), thus the discrepancies observed in the aforementioned studies may be attributed to variables such as aquatic animal species, water environment and feed composition. Nevertheless, overall evidence suggests that supplementing high-fat diets with inulin holds promise for enhancing richness and diversity within intestinal microbiota to some extent. Inulin demonstrates potential properties for maintaining bacterial equilibrium and safeguarding intestinal health.
In this study, a high-fat diet can result in reduced immune function, oxidative stress-induced damage, histopathological abnormalities in the intestines, and disruptions to the intestinal flora of spotted sea bass. Based on the findings of this study, inulin can be utilized as a dietary supplement to enhance immunity, increase antioxidant capacity, improve intestinal tissue morphology, and promote a healthy intestinal microbiota. Furthermore, inulin exhibits probiotic effects by modulating the structure of the gut microbiota with its mechanism potentially linked to microbial metabolism.
Author contribution
Fuqiang Quan: The experimental idea, Analysis, sample collection, Manuscript revision, Laboratory work, Samples analysis, Manuscript revision. Xiujuan Wang , Zhangfan Haung, Yunting Zhao, Luiming Kong, Yi Lin, Hao Lin, Sishun Zhou, Jianrong Ma, Yanbo Zhao, Longhui Liu: participated in the sample collection, discussed the result together.Zhongbao Li: conceived and designed the experiments, reviewed drafts of the paper.
Funding: Science and Technology Program of Fujian Province (Grant No. 2015N0010)， Science and Technology Program of Xiamen City (Grant No. 3502Z20143017).
Data Availability: All data in this study are available upon request.
Ethical approval: All contents of this study are subject to the Regulations on the Administration of Experimental Animals (amended on March 1, 2017 by the National Animal Control Regulations of the People's Republic of China No. 11 and Laws and Regulations of The State Council No. 676), and approved by the Animal Experiment Ethics Committee of Jimei University (Xiamen, China), approval code: JMU202103009.
Competing interests : The authors declare no competing interests.
Consent to participate : All authors and contributors involved in this research paper are readily available and accessible to provide additional information, or clarifications to anyone who may request it.
Consent for publication The authors hereby transfer the exclusive copyright of this manuscript to the journal/publisher, granting them the sole and universal right to publish, distribute, and commercialize the work in any language, format (print or electronic), and territory, without any restrictions.
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No competing interests reported.