Effects of Dietary Peanut Skin Proanthocyanidin Supplementation on Antioxidant Capacity and Intestinal Health of Juvenile American Eels (Anguilla rostrata)

TL;DR

Adding peanut skin proanthocyanidins to the diet of juvenile American eels improves their antioxidant levels and gut health, with the best results at 900 mg/kg.

Contribution

This study identifies peanut skin proanthocyanidins as a cost-effective alternative to grape seed proanthocyanidins for improving eel health.

Findings

PSPc supplementation increased antioxidant enzyme activities and reduced oxidative stress markers in eels.

Optimal intestinal health improvements were observed at 900 mg/kg PSPc, including enhanced villus height and enzyme activity.

PSPc at 900 mg/kg altered gut microbiota composition, increasing beneficial bacteria and reducing harmful ones.

Abstract

Proanthocyanidins from grape seeds are widely used as functional aquafeed additives for aquatic animals, but their high cost and limited domestic supply restrict widespread application. Peanut skins, a common agricultural byproduct, are rich in proanthocyanidins, offering a low-cost alternative resource. This study concentrates on evaluating the effects of peanut skin proanthocyanidins on the antioxidant capacity and intestinal health of juvenile American eels, and deeply analyzes the underlying mechanisms by which peanut skin proanthocyanidins enhance the antioxidant capacity and improve intestinal health of the experimental animals.

This study was performed to explore the influences of dietary peanut skin proanthocyanidins (PSPcs) on the antioxidant capability and intestinal health of juvenile American eels (Anguilla rostrata). The American eels (10.50 ± 0.03 g) were randomly allocated to five groups. Five experimental diets were formulated with graded levels of PSPc supplementation, and designated as PSPc0, PSPc300, PSPc600, PSPc900, and PSPc1200, respectively. The feeding experiment lasted for 8 weeks. In comparison with the PSPc0 group, the PSPc supplementation groups exhibited higher total antioxidant capacity and elevated levels of reduced glutathione, along with increased activities of superoxide dismutase, catalase, glutathione peroxidase, and glutathione S-transferase in the serum, liver, and intestine (p < 0.05). Dietary PSPc supplementation significantly decreased malondialdehyde levels in the serum, liver, and intestine (p < 0.05) and down-regulated hydrogen peroxide and nitric oxide levels in the liver and intestine (p < 0.05). Dietary PSPc supplementation also enhanced hepatic and intestinal anti-free radical superoxide anion and anti-free radical hydroxyl capacities (p < 0.05). Most of these parameters peaked in the PSPc900 group. PSPc supplementation in the diet increased villus height and muscular thickness and elevated activities of intestinal lipase and protease (p < 0.05), with lipase activity being the highest in the PSPc900 group. Compared with the PSPc900 group, the above-mentioned parameters in the PSPc1200 group showed a negative effect. Relative to the PSPc0 group, the PSPc900 group exhibited an increased relative abundance of Xanthomonadaceae and reduced relative abundances of Citrobacter, Chroococcidiopsis, Escherichia Shigella, Cupriavidus, Pelomonas, and Romboutsia (p < 0.05). Taken together, appropriate dietary PSPc supplementation improves antioxidant capacity and intestinal health in American eels, and the optimal dietary supplementation level is recommended at 900 mg/kg.

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1. Introduction

Proanthocyanidins are polyphenolic substances distributed in various botanical materials, including grape seeds, peanut skins, pine bark, cranberries, bayberries, and persimmons [1]. They exhibit diverse beneficial bioactivities, including antioxidant, lipid-lowering, anti-inflammatory, and antibacterial effects [2,3]. In recent years, the application of proanthocyanidins as functional aquafeed additives has attracted increasing interest, with grape seed proanthocyanidin extract (GSPE) being the most commonly studied proanthocyanidins source. Research indicates that appropriate dietary GSPE supplementation levels enhance the antioxidant capacity of multiple aquatic animals, including snakehead (Channa argus) [4], Japanese seabass (Lateolabrax japonicus) [5], common carp (Cyprinus carpio) [6], and goldfish (Carassius auratus) [7]. Meanwhile, GSPE has been reported to increase intestinal protease and lipase activity in hybrid sturgeon (Acipenser baeri Brandt ♀ × A. schrenckii Brandt ♂) [8], while also modulating the intestinal microbiota composition of snakehead [4], common carp [6], and hybrid sturgeon [8]. These biological effects of GSPE ultimately contribute to the improved growth performance of farmed fish. However, the application of GSPE is constrained by the limited domestic supply and high extraction costs of grape seed-derived proanthocyanidins in China, which necessitates large-scale imports. Consequently, there is an urgent need to identify alternative sources of proanthocyanidins.

Peanut (Arachis hypogaea L.) possesses rich germplasm resources and is widely cultivated across diverse regions [9,10]. Peanut skins account for approximately 3.5% to 4.5% of the total nut weight and can contain up to 17% proanthocyanidins [11]. Over 900,000 tons of peanut skins are produced worldwide each year, yet most are either discarded, indicating substantial untapped potential [12,13]. Research shows that dietary PSPc supplementation enhances antioxidant capacity, reduces inflammation, and modulates intestinal microbiota in mice [14,15]. PSPc can also boost cellular antioxidant capacity and exert cytoprotective effects [16,17]. Additionally, owing to peanut skins having a high concentration of PSPc, dietary inclusion of peanut skins can improve antioxidant capacity of sheep, broiler chickens, and goats [18,19,20]. Therefore, the isolation of proanthocyanidins from peanut skins represents a high-value strategy for byproduct utilization, effectively transforming agricultural waste into a functional resource. However, research on the utilization of PSPc as a functional aquafeed additive remains very limited, and its effects on the antioxidant capacity and intestinal health of farmed fish remain unclear.

Eels (Anguilla spp.) are globally important commercial fish, renowned for their high nutritional value [21]. They represent a key aquaculture product in international aquaculture trade. As a major eel farming country, China yielded approximately 310,400 metric tons from eel aquaculture in 2024 [22]. Among the eel species cultivated in China, the American eel (Anguilla rostrata) is a primary farmed species at present, with its production expanding rapidly due to its high market value and adaptability to intensive farming systems [21]. During the culturing process, eels are vulnerable to various feed-related stressors (histamine, oxidized fats and their products, etc.), which can trigger oxidative stress and compromise intestinal health, thereby impeding growth performance [23]. Therefore, dietary antioxidant supplementation is commonly implemented to alleviate these detrimental effects [24,25]. Our previous study reported that dietary PSPc supplementation improved the growth performance of American eels, possibly attributed to its ability to augment antioxidant capacity and improve intestinal health [26]. Therefore, this study selected juvenile American eels as the experimental animal to evaluate the impacts of varying levels of dietary PSPc on the antioxidant capacity, intestinal digestive enzymes, intestinal tissue morphology, and intestinal microbiota composition, which would provide meaningful references for the incorporation of PSPc into eel diets.

2. Materials and Methods

2.1. Experimental Design and Feeding Trial

The feeding trial was conducted using the same experimental design and the same batch of juvenile American eels as described in Wang et al. [26].

A commercial powdered feed for juvenile American eels (provided by Fuzhou Xinruiyi Industrial Co., Ltd., Fuzhou, China) was used as the basal diet in this trial. Its proximate composition (analyzed values) was as follows: moisture 5.44%, crude ash 14.99%, crude lipid 5.17%, and crude protein 46.14%. Five experimental diets, isonitrogenous and isolipidic, were prepared under laboratory conditions by supplementing the basal diet with powdered PSPc (purity ≥ 95%, supplied by Shandong Jinsheng Biological Technology Co., Ltd., Linyi, China) at graded concentrations of 0, 300, 600, 900, and 1200 mg/kg, and were designated as the PSPc0, PSPc300, PSPc600, PSPc900, and PSPc1200 groups, respectively.

This study used experimental fish is juvenile American eel purchased from Fujian Jinjiangzhiman Aquatic Technology Co., Ltd., Zhangzhou, China. After a 4-week adaptation period, a total of 450 unformed juvenile American eels, with an initial body weight of 10.50 ± 0.03 g (mean ± S.D.), were randomly allocated to 15 circular PVC tanks (400 L of water in each tank). Each tank contained 30 eels, with three replicates per group. The 8-week formal feeding trial was carried out in a recirculating aquaculture system, with a continuous water flow rate of 2.0 L/min per tank. The system was equipped with a temperature control system and air stones to maintain constant water temperature and dissolved oxygen level, and the entire trial was performed under a continuous dark environment throughout the experimental period. Before feeding, water was added to the powdered diet in a 1:1.1 ratio to create experimental diets with a consistent dough-like texture. Eels were hand-fed twice daily at 06:00 and 18:00, with an initial single feeding rate of 0.1% body weight. The feeding rate was adjusted stepwise: if feed was completely consumed 30 min post-feeding, the next meal’s feeding rate was increased by 0.1% body weight, until apparent satiation was reached. Uneaten feed was collected with a net at 30 min post-feeding, dried at 65 °C to a constant weight, and accurately weighed to calculate the actual daily feed intake of each tank. Water quality was monitored and kept within the following ranges: temperature 25.00–26.10 °C, pH 7.00–7.80, dissolved oxygen 8.10–9.50 mg/L and total ammonia nitrogen < 0.25 mg/L.

2.2. Sample Collection

After the conclusion of the 8-week formal feeding trial, the final body weight of the experimental eels ranged from 16.10 to 19.70 g/fish. As dietary PSPc levels increased, final body weight and weight gain rate first increased and then decreased in juvenile American eels, with the highest values observed in the PSPc900 group. All American eels were subjected to a 24-h fasting period before being sampled. According to the anesthetic concentration used for eel sampling in previous studies, fifteen eels per tank were randomly selected to be anesthetized with 0.1 g/L eugenol [26]. Blood samples were collected and kept at 4 °C for 24 h before centrifugation (4 °C, 3500 r/min, 10 min). The resulting supernatant serum was stored at −80 °C. The eels were dissected with sterile scissors to remove the visceral mass, from which the intestine and liver were separated and rinsed with 0.01 M phosphate-buffered saline (PBS, pH 7.4). Intestine and liver samples from six eels per tank were collected to assess enzyme activities and antioxidant capacity. Six eels per tank were sampled for intestinal microbiota composition analysis. Additionally, intestinal samples from three eels per tank were collected for histological examination. All intestinal samples were collected from the midgut, with sterile dissection operation for microbiota samples, immediate 4% paraformaldehyde fixation for histological samples, and snap-freezing in liquid nitrogen for biochemical samples.

2.3. Determination of Antioxidant Capacity

Liver and intestinal tissues from six eels per tank were homogenized using a tissue homogenizer (Tissuelyser-24, Shanghai Jingxin Industrial Development Co., Ltd., Shanghai, China) in pre-cooled sterile normal saline at a mass-to-volume ratio of 1:9 (g/mL), followed by centrifugation at 3500 rpm for 10 min at 4 °C. The supernatants were then collected and pooled into one composite sample for subsequent assays. Serum samples could be directly subjected to subsequent detection without pre-treatment. All operations were performed on ice to prevent the degradation of enzyme activity.

The total antioxidant capacity (T-AOC, colorimetric method, #A015-1-2), the activities of superoxide dismutase (SOD, hydroxylamine method, #A001-1-1), catalase (CAT, ammonium molybdate method, #A007-1-1), glutathione peroxidase (GSH–Px, colorimetric method, #A005-1-2) and glutathione g-transferase (GST, colorimetric method, #A004-1-1), the levels of reduced glutathione (GSH, colorimetric method, #A006-1-1) and malondialdehyde (MDA, thiobarbituric acid method, #A003-1-2), and the activities of anti-free radical superoxide anion (ASA, colorimetric method, #A052-1-1) and anti-free radical hydroxyl ability (AHA, colorimetric method, #A018-1-1), along with hydrogen peroxide (H_2_O_2_, colorimetric method, #A064-1-1) and nitric oxide (NO, nitrate reductase method, #A013-2-1) levels, were measured with the use of commercial kits (Nanjing Jiancheng Biotechnic Institute, Nanjing, China). All assays were performed strictly according to the manufacturer’s instructions, with absorbance detected by a multifunctional microplate reader (Infinite 200pro, Tecan Austria GmbH, Grödig, Austria).

2.4. Intestinal Histological Observation

Intestinal H&E-stained sections were produced in accordance with the procedure outlined by Zhai et al. [27]. Intestinal tissues from two eels per tank were fixed in Bouin’s solution for 24 h, cut into appropriate segments, and dehydrated in a fully automatic tissue dehydrator (Donatello, Diapath, Milan, Italy). After embedding with a tissue embedding machine (JB-P5, Wuhan Junjie Electronic Co., Ltd., Wuhan, China), 4 μm-thick serial sections were cut using a rotary paraffin microtome (RM2016, Leica Microsystems Co., Ltd., Shanghai, China). The sections were processed on a tissue flotation machine (Jinhua Kedee Instrument Equipment Co., Ltd., Jinhua, China), air-dried at 60 °C, and stained with H&E. Afterward, the sections were examined under a microscope (BX80-JPA, Olympus, Tokyo, Japan) to capture representative images. Morphometric measurements were performed with the utilization of Image-Pro Plus 6.0 software (National Institutes of Health, Bethesda, MD, USA).

The processing of American eel intestinal samples for scanning electron microscopy was carried out according to the method described by Allam et al. [28]. Intestinal tissue from one eel per tank blocks (3 mm^2^) were rinsed with PBS to remove surface contaminants, fixed in SEM fixative for 2 h at room temperature and then stored at 4 °C in the dark, and rinsed again with PBS. The tissues were subsequently dehydrated through a graded series of ethanol, acetone, and isoamyl acetate, dried in a critical point dryer (K850, Quorum, Laughton, UK), and sputter-coated with gold for 30 s using an ion sputter coater (MC1000, Hitachi, Tokyo, Japan). The prepared samples were then examined and imaged under a scanning electron microscope (SU8100, Hitachi, Tokyo, Japan).

2.5. Analysis of Intestinal Digestive Enzymes

The pre-treatment of intestinal samples was conducted as described in Section 2.3. The activities of digestive enzymes, including amylase (starch-iodine colorimetric method, #C016-1-1), lipase (methylresorufin substrate method, #A054-2-1) and protease (Folin-phenol method, #A080-1-1), were analyzed by the kits (Nanjing Jiancheng Biotechnic Institute). All assays were performed strictly according to the manufacturer’s instructions, with absorbance detected by a multifunctional microplate reader (Infinite 200pro, Tecan Austria GmbH, Austria).

2.6. Analysis of Intestinal Microbiota

Due to the PSPc900 group exhibiting the best growth performance in the feeding trial reported in the study of Wang et al. [26], the PSPc0 and PSPc900 groups were selected for further intestinal microbiota analysis. Intestinal microbiota profiling analysis was carried out referring to the procedure of Xu et al. [29]. For this analysis, six intestinal samples per tank were pooled to form two composite samples (three individuals per composite sample). With three replicate tanks per group, a total of six composite samples were analyzed for each group. Total DNA was isolated from all intestinal samples. The quality of extracted DNA was verified by 1% agarose gel electrophoresis. Qualified samples were subsequently subjected to high-throughput sequencing focusing on the V3–V4 region of the bacterial 16S rRNA gene on an Illumina MiSeq PE300 platform (Beijing Ovison Gene Technology Co., Ltd., Beijing, China).

2.7. Statistical Analysis

One-way ANOVA was conducted using SPSS 26.0 (SPSS, Chicago, IL, USA), with Duncan’s method for subsequent multiple comparison analysis. Statistical significance was set at p < 0.05. The data were expressed as mean ± standard deviation (S.D., n = 3 tanks). The raw sequencing data were filtered to obtain quality-filtered reads, which were clustered into operational taxonomic units (OTUs). Alpha-diversity indices of the intestinal microbiota were quantified using QIIME software (version 1.9.0). Beta-diversity was assessed by PLS-DA. Python software version 2.7 was employed for Lefse analysis, applying parameters of an LDA score cutoff of 2 and p < 0.05 to identify differentially abundant bacterial taxa between groups.

3. Results

3.1. Antioxidant Capacity in Serum, Liver, and Intestine

Antioxidant capacity of juvenile American eels in various PSPc groups is presented in Figure 1, Figure 2 and Figure 3. Compared to the PSPc0 group, the PSPc supplementation groups exhibited significantly enhanced levels of T-AOC and GSH, as well as significantly elevated activities of SOD, CAT, GSH-Px, and GST in the serum, liver, and intestine (p < 0.05). These parameters were significantly higher in the PSPc900 group compared to the other treatments (p < 0.05), and they initially increased and then decreased with increasing dietary PSPc levels. Additionally, dietary PSPc supplementation significantly reduced MDA levels in the serum, liver, and intestine. As dietary PSPc supplementation levels increased, MDA levels initially decreased and then increased, with the lowest levels observed in the PSPc900 group.

As shown in Figure 4, compared with the PSPc0 group, PSPc supplementation groups showed significantly higher activities of intestinal and hepatic ASA and AHA (p < 0.05). These parameters showed initially increased and then decreased with increasing dietary PSPc levels, with the highest overall levels observed in the PSPc900 group. The activities of hepatic ASA and AHA were significantly higher in the PSPc900 group compared to the other treatments (p < 0.05). Dietary PSPc supplementation significantly decreased levels of H_2_O_2_ and NO in the liver and intestine (p < 0.05). As the dietary PSPc level increased, the levels of hepatic and intestinal H_2_O_2_ and NO initially decreased and then increased, reaching their lowest levels in the PSPc900 group.

3.2. Intestinal Morphology

As shown in Figure 5, in comparison with the PSPc0 group, the PSPc600, PSPc900, and PSPc1200 groups exhibited significantly increased intestinal villus length and muscular thickness in American eels (p < 0.05). The intestinal villus length and muscular thickness showed a pattern of initial increase followed by a decrease with increasing PSPc levels, peaking in the PSPc600 and PSPc900 groups, respectively. PSPc supplemented groups exhibited more distinct intestinal cell boundaries and increased microvillus density in comparison with the PSPc0 group, with the PSPc900 group showing the most distinct boundaries and the highest microvillus density.

3.3. Intestinal Digestive Enzymes Activities

As shown in Figure 6, compared to the PSPc0 group, the PSPc supplementation groups significantly increased the intestinal activities of lipase and protease (p < 0.05). Lipase activity was significantly higher in the PSPc900 group compared to the other treatments, and it initially increased and then decreased with increasing PSPc levels. There was no significant difference in intestinal amylase activity among all groups (p < 0.05).

3.4. Intestinal Microbiota

As shown in Figure 7A–D, there were no significant differences in the alpha diversity, including chao1 index, observed species, PD whole tree index, and Shannon index, of the intestinal microbiota between eels fed these two diets. PLS-DA analysis revealed clear separation between the PSPc0 and PSPc900 groups, with tight clustering within each group, indicating distinct intestinal microbial communities (Figure 7E). At the phylum level, the dominant intestinal microbiota in both groups primarily consisted of Tenericutes, Firmicutes, Proteobacteria, Actinobacteria, and Fusobacteria. There were no significant differences in microbial composition at the phylum level between the two groups (p < 0.05) (Figure 7F). Lefse analysis indicated that, compared with the PSPc0 group, the PSPc900 group exhibited a higher relative abundance of Xanthomonadaceae (p < 0.05) and lower relative abundances of Citrobacter, Chroococcidiopsis, Escherichia Shigella, Cupriavidus, Pelomonas, and Romboutsia (p < 0.05) (Figure 7G).

4. Discussion

4.1. Antioxidant Capacity in Serum, Liver, and Intestine

The fish antioxidant defense system relies on pivotal functions mediated by antioxidant enzymes (SOD, CAT, GSH-Px, GST) and the antioxidant substance GSH [4]. Specifically, SOD and CAT possess the ability to decompose free radicals stepwise [30,31]. GSH-Px utilizes GSH to decompose H_2_O_2_ and lipid peroxides [30], and GST acts with GSH-Px to eliminate lipid peroxides [31]. The thiol group of GSH can directly scavenge oxygen radicals and inhibit lipid peroxidation [6]. T-AOC can evaluate the antioxidant system in fish [32]. MDA directly reflects the extent of lipid peroxidation and endogenous oxidative damage [5]. In the present study, the activities of SOD, CAT, GSH-Px, and GST, as well as the concentrations of GSH and T-AOC, were significantly elevated in the serum, liver, and intestine of juvenile American eels following dietary PSPc supplementation, while MDA content was significantly diminished in these tissues. With increasing dietary PSPc levels, all these parameters (except MDA) showed an initial increase, then decrease, whereas MDA levels exhibited an opposite trend. The optimal values for these indicators were observed in the PSPc900 group. Our results correspond to investigations demonstrating that appropriate dietary levels of PSPc increased SOD activity and reduced MDA levels in the intestines of ulcerative colitis mice [14], mouse hepatocytes [33], mouse embryonic fibroblast cells [17], and human liver cells [34]. Furthermore, dietary supplementation with peanut skins, which contain PSPc, was shown to enhance the activities of CAT and SOD in broiler muscle [20] and to decrease serum MDA levels in goats [18]. Taken together, the alterations in these antioxidant indices indicate that appropriate dietary PSPc supplementation can augment the overall antioxidant capacity of experimental animals.

The common intracellular free radicals primarily include the superoxide anion (O_2_^−^), hydroxyl radical (·OH), hydrogen peroxide (H_2_O_2_), nitric oxide (NO), etc. Among them, O_2_^−^, ·OH, and H_2_O_2_ are classified as reactive oxygen species (ROS), and NO is classified as a reactive nitrogen species (RNS). Excess free radicals can damage cell membrane structure, reduce antioxidant enzyme activity, provoke inflammatory responses, and induce cell apoptosis [35]. The levels of O_2_^−^ and ·OH can be, respectively, evaluated by the indicators ASA and AHA [36]. In this experiment, as dietary PSPc levels increased, the values of intestinal and hepatic ASA and AHA in juvenile American eels exhibited an initial rise followed by a decline. This trend indicates that levels of O_2_^−^ and ·OH were initially decreased and then increased, and the levels of H_2_O_2_ and NO followed a comparable pattern. It was reported that appropriate dietary PSPc supplementation can reduce intestinal NO levels in ulcerative colitis mice and decrease ROS levels in mouse embryonic fibroblasts and human liver cells [14,17,34]. This evidence indicates that optimal dietary PSPc supplementation reduces free radical levels in the liver and intestine of experimental animals, further demonstrating an enhancement in their antioxidant capacity.

Based on the existing research, appropriate dietary PSPc supplementation improves the antioxidant ability of experimental animals through elevating the content of antioxidant substances and the activities of antioxidant enzymes, along with decreasing free radical levels in the liver and intestine. It has been reported that PSPc has metal-chelating activity, chelating metal ions that mediate free radical generation, thus inhibiting radical production [37]. PSPc is also able to improve the transcriptional level of antioxidant genes and activate antioxidant-related signaling pathways [33,38]. PSPc incorporates into the cell membranes, maintaining membrane function while moderately reducing membrane fluidity, thereby inhibiting lipid peroxidation [8,39]. PSPc decreases the level of inflammatory cytokines and NADPH oxidase activity by inhibiting the nuclear factor-κB pathway, thereby reducing ROS generation [40]. PSPc induces autophagy, clears oxidatively damaged cellular components, maintains intracellular homeostasis, and prevents ROS generation from impaired proteins and organelles [17]. Thus, PSPc comprehensively enhances the body’s antioxidant capacity by chelating metals, activating endogenous antioxidant pathways, preserving membrane integrity, inhibiting inflammatory signals, and inducing autophagy.

In addition, relative to the PSPc900 group, the PSPc1200 group displayed a certain weakening effect on the antioxidant capacity. This phenomenon may be ascribed to PSPc, as a polyphenol, which is prone to auto-oxidative polymerization at high doses, thereby weakening its reducing capacity. Moreover, its failure to activate energy metabolism pathways impairs the synthesis and function of antioxidant enzymes [41,42].

4.2. Intestinal Morphology

As the principal site of nutrient absorption in fish, the intestine exhibits increased muscular layer thickness that strengthens contractile force to enhance digestion, while increased villus length and density expand the absorptive surface area and improve absorption efficiency [43,44]. In the present trial, dietary PSPc supplementation exceeding 600 mg/kg enhanced the intestinal villus length, muscular thickness, and microvillus density of juvenile American eels, while American eels fed a diet containing 900 mg/kg PSPc exhibited the optimal improvement in intestinal morphology. These results are consistent with studies reporting that appropriate dietary PSPc supplementation promotes intestinal mucosal repair in ulcerative colitis mice [14]. Consequently, appropriate dietary PSPc supplementation can improve intestinal morphology in experimental animals, potentially through regulating intestinal microbiota, optimizing bile acid metabolic profiles, and reducing intestinal inflammation [14]. Furthermore, high dietary PSPc supplementation may weaken intestinal morphological improvement, possibly because it exerts a broad-spectrum inhibitory effect, which decreases microbial diversity and alters bile acid metabolism [14].

4.3. Intestinal Digestive Enzymes Activities

The activity of digestive enzymes, a key determinant of nutrient digestion and absorption, is crucial for growth performance in fish. Dietary lipids and proteins are hydrolyzed into absorbable small molecules by the catalytic actions of intestinal lipase and protease, respectively [45]. In this study, dietary PSPc supplementation significantly enhanced the activities of intestinal lipase and protease. With the increase in dietary PSPc levels, lipase activity exhibited an initial increase followed by a decrease, and its peak activity was achieved in the PSPc900 group. The impact of PSPc on digestive enzyme activities has not been previously reported. It was only demonstrated that optimal dietary GSPE supplementation augments intestinal lipase and protease activities in hybrid sturgeon [8]. Our findings indicate that optimal dietary PSPc supplementation can enhance intestinal digestive enzyme activity in American eels. The mechanism may be analogous to that of GSPE, modulating intestinal microbiota and improving intestinal morphology, which provides a conducive environment for enzymatic activity [4,8,46]. Additionally, high dietary PSPc supplementation reduced intestinal lipase activity in American eels, possibly because it decreased intestinal microbiota diversity and diminished beneficial effects on intestinal morphology [14]. Notably, amylase activity was not significantly affected by PSPc supplementation in the present study. This may be attributed to the carnivorous nature of American eels, in which intestinal amylase activity is relatively stable.

4.4. Intestinal Microbiota

The fish intestine hosts a complex and symbiotic ecosystem of microbiota. The structure of intestinal microbiota is subject to multiple influences, including developmental stage, nutritional status, and diet, and it exerts crucial functions in processes such as immune modulation and intestinal health maintenance [47,48]. PLS–DA analysis revealed significant differences in the intestinal microbiota between the PSPc0 group and PSPc900 group, indicating that dietary PSPc supplementation altered intestinal microbiota in American eels. It has been reported that PSPc exerts a selective modulatory effect on the intestinal microbiota, enhancing the proportion of beneficial bacteria and suppressing harmful bacteria, thereby altering microbial diversity [14]. Lefse analysis indicated that the PSPc900 group had an increased level of Xanthomonadaceae and reduced levels of Citrobacter, Chroococcidiopsis, Escherichia Shigella, Cupriavidus, Pelomonas, and Romboutsia. Research indicates that some members of Xanthomonadaceae can produce a range of digestive enzymes, including cellulases, which may promote nutrient digestion and absorption [49]. Some strains of Xanthomonadaceae not only antagonize pathogenic fungi and nematodes but also break down deleterious organic compounds in aquaculture systems [50,51]. The genus Citrobacter is a facultatively anaerobic, Gram-negative bacterium frequently identified as a pathogen in fish [52]. Infection with Citrobacter freundii triggers the innate mucosal immune system and induces acute intestinal inflammation in grass carp [53]. Citrobacter freundii is also capable of causing hemorrhagic septicemia in fish [54,55]. Chroococcidiopsis was found in the sediments of ponds for Nile tilapia [56], but its role within the fish intestine remains unreported. Escherichia Shigella is one of the opportunistic pathogens in aquatic animals, which can cause intestinal diseases under specific conditions [57,58]. Research has indicated that appropriate dietary PSPc supplementation can significantly reduce the level of Escherichia Shigella in the mouse intestine [59], which corresponds to the findings of our study. Cupriavidus is associated with the metabolism and homeostasis of certain metal elements and serves as a dominant bacterial genus in the intestines of fish, including Glyptosternum maculatum and large yellow croaker (Larimichthys crocea) [60,61]. As a pathogen, Cupriavidus is frequently present in individuals with weakened immunity, whose relative abundance in the intestine correlates positively with the level of pro-inflammatory factors [62]. Pelomonas has been detected in the intestine of fish, such as cobia (Rachycentron canadum), milkfish (Chanos chanos), and zebrafish (Danio rerio) [63,64]. It was reported that Pelomonas is a dominant bacterium in patients with severe intestinal inflammation [65]. Romboutsia is a thermophilic anaerobic bacterium, which has been detected in the intestine of channel catfish (Ictalurus punctatus) and is associated with glycometabolism in tilapia [66,67]. Therefore, our experimental results indicated that dietary supplementation with 900 mg/kg PSPc exerted a selective modulatory effect on the intestinal microbiota of juvenile American eels, characterized by both the enhancement of beneficial bacteria and the suppression of potentially pathogenic genera. As a type of proanthocyanidin, PSPc may modulate intestinal microbiota selectively through multiple complementary mechanisms. The polyphenolic components of PSPc can directly disrupt bacterial cell membrane integrity and interact with intracellular macromolecules, with Gram-negative pathogens being particularly susceptible due to their outer membrane structure [68,69]. PSPc may function as a prebiotic substrate, selectively stimulating the growth of beneficial bacteria that possess the enzymatic capacity to metabolize polyphenolic compounds [14,70]. By promoting beneficial bacteria, PSPc indirectly suppresses pathogens through competitive exclusion for nutrients and adhesion sites, as well as through the production of antimicrobial metabolites by the enriched beneficial taxa [71]. Proanthocyanidins can interfere with bacterial quorum sensing and virulence factor expression, thereby attenuating pathogenicity [72]. Collectively, these mechanisms account for the observed bidirectional regulation: promoting beneficial bacteria with adaptive metabolic capacities while inhibiting potential pathogens.

From the above results, the PSPc900 group was significantly superior to the other PSPc supplementation groups in antioxidant indices, free radical levels, intestinal morphology, and digestive enzyme activities of American eels. Meanwhile, the intestinal microbiota in the PSPc900 group was beneficially regulated. Therefore, the PSPc900 group showed the highest ability to improve antioxidant capacity and intestinal health, which was consistent with the optimal level of PSPc supplementation at 900 mg/kg to promote growth reported in our previous study [26].

5. Conclusions

In summary, this study demonstrated that an appropriate level of dietary PSPc supplementation elevated the antioxidant capacity in the serum, liver, and intestine, improved intestinal morphology, and increased intestinal protease and lipase activities in juvenile American eels. Furthermore, dietary supplementation with 900 mg/kg PSPc decreased the abundance of potentially pathogenic bacteria and increased that of beneficial bacteria in the intestine of juvenile American eels. Thus, our results provide meaningful insights for the application of PSPc in American eels and recommend an optimal dietary supplementation level of 900 mg/kg.