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. 2025 Nov 26;11:100331. doi: 10.1016/j.fochms.2025.100331

Glutathione and astaxanthin: Both enhance antioxidant capacity and regulate lipid metabolism in Pacific white shrimp (Litopenaeus vannamei) fed with high-lipid diets

Jiandong Chen a, Tao Cheng a, Xiang Si a, Fan Yang b, Jianhua Yi b, Zhilong Yang b, Zhaowen Li b, Beiping Tan a, Chunfeng Yao c,⁎, Shuyan Chi a,⁎
PMCID: PMC12719031  PMID: 41438199

Abstract

Glutathione (GSH) and astaxanthin (AX) have the function of helping lipid metabolism and antioxidant capacity; which one is better? The feeding experiment of an 8 weeks was conducted to investigate the advantages of these two working on Litopenaeus vannamei fed with high-lipid diets. Four diets were formulated: basal diet (CON, 9 % lipid content); high-lipid diet (HL, 11 % lipid content); and supplementation with 6.7 g/kg Glutathione-rich yeast hydrolysate (GSH, 5 %) and 0.3 g/kg CarophyllⓇ Pink (AX, 10 %) based on the HL diet, recorded as HLG and HLA, respectively. The results demonstrated a clear distinction between these two supplements. Compared to the HL group, shrimp in the HLG and HLA groups exhibited a consistent pattern of improvement that significantly reduced hepatic triglyceride levels, markedly downregulated expression of the fatty acid synthase (fas) gene, and enhanced activities of the key antioxidant enzymes catalase and glutathione peroxidase. Compared to the HLA group, the HLG group exhibited upregulation of triacylglycerol lipase (tgl) and glucose-6-phosphatedehydrogenase (g6pdh) gene expression and lower TG content. Shrimp fed HLG diet exhibited superior overall performance, maintaining growth at levels equivalent to the control (CON) group. Specifically, the HLG group showed significantly greater thickness of intestinal muscular layer, and height and width for intestinal villus compared to both the HL and HLA groups (P < 0.05). In summary, while both additives mitigated hepatic lipid accumulation and oxidative stress, GSH delivered stronger benefits for growth performance through regulating antioxidant capacity and intestinal health when shrimp fed high-lipid diet.

Keywords: Glutathione, Astaxanthin, High lipid diet, Antioxidant capacity, Lipid metabolism, Shrimp

Highlights

  • •

    Both glutathione and astaxanthin reduced hepatic triglyceride in shrimp.

  • •

    Both glutathione and astaxanthin boosted key antioxidant capacity in shrimp.

  • •

    Glutathione improved the growth and intestinal health in shrimp.

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    Glutathione uniquely upregulated lipid catabolism tgl gene expression levels.

1. Introduction

The rising cost of conventional protein sources, particularly fishmeal, has imposed significant economic constraints on the sustainability of aquaculture (Aragão et al., 2022). To address this issue, the “protein-sparing effect” has been brought to the forefront and is receiving more attention. Lipids serve as essential nutrients in aquafeeds, fulfilling multiple physiological functions: they are integral components of cell membranes, facilitate the absorption and transport of lipid-soluble vitamins, provide a dense source of energy, and enhance overall feed utilization, thereby reducing the reliance on protein and contributing to protein conservation (Turchini et al., 2009; Zheng et al., 2022). However, crustaceans, including Pacific white shrimp (Litopenaeus vannamei), face inherent challenges in lipid digestion and utilization due to their physiological limitations. The lipid digestive physiology of crustaceans differs fundamentally from that of vertebrates (Li et al., 2023). Although crustaceans possess endogenous emulsifiers (such as fatty acylsarcosyltaurine, fatty acyl-taurine, and fatty acyl-dipeptide) (Holwerda & Vonk, 1973; Van Den Oord et al., 1965), their overall emulsification system is less efficient than the bile acid-dependent system of vertebrates (Su et al., 2022). When the lipid content in shrimp feed is relatively high, the shrimp's ability to efficiently utilize the feed lipids becomes challenging. Several negative effects have been observed in Litopenaeus vannamei, including impaired growth performance (Zhang et al., 2013b), oxidative stress (Xu et al., 2018), excessive lipid deposition (Hamidoghli et al., 2020), and lipid peroxidation (Raghuvaran et al., 2023). These effects collectively lead to excessive production of reactive oxygen species (ROS), causing oxidative damage, organelle dysfunction, and ultimately cell death (Dai et al., 2019; Liu et al., 2022b; Marra & Svegliati-Baroni, 2018). Therefore, it is necessary to develop strategies that alleviate these adverse effects and enhance lipid utilization efficiency for shrimp fed high-lipid diets.

The popular name for 3, 3′-dihydroxy-β, β’-carotene-4, 4′-dione is Astaxanthin (AX), commonly applied as an antioxidant in aquaculture nutrition (Lim et al., 2018). The unique molecular structure of AX, characterized by hydroxyl and ketone groups, allows it to quench singlet oxygen and neutralize free radicals, thereby inhibiting lipid peroxidation and protecting cellular membranes from oxidative damage (Brotosudarmo et al., 2020; Nair et al., 2023). Dietary AX supplementation could improve lipid metabolism, immune response, oxidative stress, and inflammation in crustaceans (Deng et al., 2024; Li et al., 2022; Wang et al., 2018c; Wang et al., 2018a; Zhao et al., 2022). Meanwhile, dietary AX at 24.2 and 45.8 mg/kg significantly regulated lipid metabolism and improved the immune and antioxidant capacity in hemolymph of swimming crab (Portunus trituberculatus) (Deng et al., 2024). For Litopenaeus vannamei, dietary AX supplementation could promote shrimps to grow fast, and antioxidant ability and resistance to Vibrio harveyi infection (Eldessouki et al., 2022; Liu et al., 2022b; Zhang et al., 2013b), and modulate lipid metabolism-related gene expression (Chen et al., 2025).

Another promising antioxidant is glutathione (GSH), a bioactive tripeptide and a key intracellular antioxidant (Townsend et al., 2003). GSH exists in oxidized (GSSG) and reduced (GSH) forms in body and functions as a central regulator of cellular redox balance (Dwivedi et al., 2020; Lu, 2013). The addition of glutathione to feed has been demonstrated to promote growth, antioxidant capacity, immunity, and gut health in Litopenaeus vannamei (Luque-Ceballos et al., 2023; Wang et al., 2018b; Xia & Wu, 2018).

The study evaluated the efficacy of antioxidants between AX and GSH for Litopenaeus vannamei. By comparing and evaluating their differences in antioxidant defense capabilities and involvement in lipid metabolism, the most suitable antioxidant for high-lipid diets can be identified to improve shrimp health.

2. Materials and methods

2.1. Experimental diets

The control group used a basal diet and 2 % fish oil was increased to the basal diet as a high lipid diet (HL). The other two diets were prepared by supplementing GSH and AX separately in the HL diet, designated as HLG and HLA, respectively. Based on a combination of suitable dosages reported by previous studies and feed cost (Chen et al., 2023; Eldessouki et al., 2022; Zhang et al., 2013a; Zhao et al., 2022), 0.3 g/kg CarophyllⓇ Pink (AX, 10 %) and 6.7 g/kg Glutathione-rich yeast hydrolysate (GSH, 5 %) were supplemented separately to high-lipid diets. Feed ingredients were ground and passed through an 80-mesh sieve. All ingredients were then thoroughly blended and pelletized into 1.5-mm and 1.0-mm diameters. The pellets were dried in an air-conditioned room (25 °C) with light-blocking for 3 days. And then sealed in airtight bags and stored at −20 °C until consumption. The test diets formulation and nutrient contents were shown in Table S1.

2.2. Feeding experience

The shrimp feeding trial was carried out in the indoor culture facility. Shrimp larvae, obtained from Zhanjiang Hisenor Marine Biotechnology Co., Ltd., were fed commercial feed (Guangdong Yuehai Feed Group Co., Ltd., China) and cultured in an outdoor concrete pond for 4 weeks prior to the feeding trial.

Shrimp were fasted for 24 h, and then vigorous juvenile shrimp (initial weight 0.36 ± 0.01 g) were randomly divided into four groups in triplicate (40 shrimp per tank). The test lasted 8 weeks, with feed administered at 7:00, 11:30, 16:30, and 21:00 daily. The initial feeding regime comprised 10 % of the total biomass per day, adjusted thereafter based on consumption rates and residual feed observations. During the feeding trial, oxygen was continuously introduced into the water column. And dissolved oxygen in the water column was no less than 5 mg/L. In addition, salinity was 28–30 g/L, the temperature of water in the tank during the feeding test was 28–31 °C, ammonia nitrogen concentration was below 0.03 mg/L, and pH value was 7.7–8.2.

2.3. Sample collection

At the end of the 8-week feeding trial, all treatment groups of shrimp were subjected to a 24-h fasting period. Shrimp from each tank were enumerated, measured, and weighed. Subsequently, ten shrimps from each tank were then randomly selected for hemolymph collection. Hemolymph was aspirated from the pericardial cavity using sterile 1 mL syringes and transferred to microcentrifuge tubes. Then the hemolymph samples were kept at 4 °C for 12 h to allow clotting, followed by centrifugation at 3000 ×g for 10 min to isolate serum. The serum samples were immediately stored at a temperature of −80 °C for subsequent relative index determination.

The hepatopancreas were dissected from four shrimps per tank (12 individual samples per treatment). Each sample was placed in a sterile Eppendorf tube and stored at −80 °C until analysis for immune and antioxidant enzyme activities. For gene expression analysis, hepatopancreas tissues from two shrimp were stabilized in RNAlater at −80 °C. Hepatopancreas and intestinal tissue were separately dissected from three shrimp per tank and fixed in neutral buffered 4 % formaldehyde, and processed for histopathology. Additionally, three shrimp were randomly collected from each tank, individually placed into pre-labeled ziplock bags, and stored at −20 °C until compositional analysis. The proximate composition of whole body and diets were tested. Crude protein was determined using a Dumas nitrogenanalyzer (SNC-100, Skalar, theNether lands), crude lipid was determined by Soxhlet apparatus with petroleum ether. The crude ash was determined by low-temperature carbonization followed by calcination in a Mufflefurnace at 550 °C.

2.4. Growth and survival calculations

The growth performance parameters were computed using the following formulas (Chen et al., 2025):

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    Weight gain rate (WGR, %) = 100 × (final body weight (g) - initial body weight (g)) / initial body weight (g);

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    Survival rate (SR, %) = 100 × (final shrimp number / initial shrimp number);

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    Specific growth rate (SGR, %/day) = 100 × (Ln final weight (g) - Ln initial weight (g) / days of the experiment;

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    Feed conversion rate (FCR) = feed consumed (dry weight, g) / weight gain (g);

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    Condition factor (g/cm3) = body weight(g)/body length (cm3) × 100.

2.5. Biochemical analyses

Enzyme activity and biochemical parameters were measured using commercially available kits (Nanjing Jiancheng Institute of Biological Engineering, China). These parameters included alanine aminotransferase (ALT) aminotransferase (AST), acid phosphatase (ACP), alkaline phosphatase (AKP), catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), phenoloxidase (PO), total protein (TP), total cholesterol (T-CHO), triglyceride (TG), glucose (GLU), malondialdehyde (MDA), reduced glutathione (GSH), and total antioxidant capacity (T-AOC).

2.6. Hepatopancreas and intestinal histology

The intestines and hepatopancreas were dehydrated through a graded ethanol series: 75 % (4 h), 85 % (2 h), 90 % (2 h), 95 % (1 h), elution by anhydrous ethanol for 30 min, and repeat this step one time, alcohol-benzene for 10 min, and xylene for 10 min, repeat this step one time. Subsequently, tissues were infiltrated with molten paraffin at 65 °C in three successive stages of 1 h each. The tissues were then embedded in paraffin to form solid wax blocks. These blocks were serially sectioned into 5 μm thick slices and stained with hematoxylin and eosin. The stained sections were examined under a microscope (Nikon ECLIPSE Ni-E, Tokyo, Japan) and photographed by image acquisition software (NIS Elements version 4.60, Nikon, Japan).

Villus width (VW), villus height (VH), and intestinal wall thickness (IWT) were measured in ten random microscope fields selected to measure each intestine slices (Ray et al., 2020). The average of ten fields of view for each shrimp was calculated. The number of different cell types in five hepatic tubules per hepatopancreas slice was counted.

2.7. Real-time quantitative qRT-PCR analysis of gene expression

Total RNA was extracted from shrimp hepatopancreas using TRIzol reagent (TransGen Biotech, China), following the instructions of manufacturer. Subsequently, total RNA was reverse-transcribed into cDNA using the Evo M-MLV RT Mix Kit with gDNA Clean (AG11728, Accurate Biotechnology, Hunan, Co., Ltd). Quantitative real-time PCR (qPCR) was then performed using the SYBR Green Premix Pro Taq HS qPCR Kit II (AG11702, Accurate Biotechnology, Hunan, Co., Ltd) on a quantitative PCR instrument (Roche LightCycler® 480). Gene ef-1α was set as a reference gene. Gene expression levels were calculated by the 2-ΔΔCT method (Yang et al., 2020). The primers of genes for the RT-PCR analyses were listed in Table S2.

2.8. Statistical analysis

All original data were statistically verified using one–way analysis of variance (ANOVA). Tukey's test was used for comparing mean values between treatments. All statistics were conducted using the SPSS 21.0 (SPSS Inc., Chicago, IL, USA) at a level of P < 0.05. Data were presented as means ± standard error (SE). Experimental result figures were created by GraphPad Prism 8.0.2.

3. Results

3.1. Growth performance

There are no differences occurred in FCR, SR, and CF among all treatments (P > 0.05). Obviously, shrimps fed the HL diet got the lower FBW, WGR, and SGR compared to the CON group (P < 0.05). When shrimps ingested the HLG and HLA diets, the growth performance was improved, while the HLG group reached a level similar to that of the CON group (P > 0.05; Table 1).

Table 1.

Effect of GSH and AX supplement in high-lipid diets on growth performance of shrimp.

Index
 Diets
CON HL HLG HLA
FBW (g) 13.36 ± 0.06b 12.83 ± 0.12a 13.08 ± 0.11ab 12.91 ± 0.09a
WGR (%) 3569.56 ± 16.10b 3426.24 ± 34.14a 3494.93 ± 27.04ab 3448.76 ± 23.62a
SGR (%/d) 6.43 ± 0.01b 6.36 ± 0.02a 6.40 ± 0.01ab 6.37 ± 0.01a
FCR 1.09 ± 0.00 1.11 ± 0.01 1.08 ± 0.01 1.09 ± 0.00
SR (%) 99.17 ± 0.83 98.33 ± 0.83 97.50 ± 2.50 96.67 ± 1.67
CF (g/cm3) 0.71 ± 0.01 0.67 ± 0.01 0.69 ± 0.01 0.71 ± 0.02
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Data are expressed as means ± SE. Different lowercase letters indicate significant differences (P < 0.05).

3.2. Proximate composition of whole body

The approximate composition of whole shrimp is shown in Table S3. Moisture and crude lipid contents showed no significant differences among groups (P > 0.05). Crude protein content was similar in the HL, HLA, and HLG groups (P > 0.05). Crude ash content was significantly higher in the HL, HLA, and HLG groups compared to the CON group (P > 0.05).

3.3. Serum biochemical parameters

It can be seen from Fig. 1 that serum GLU, TG, MDA, T-CHO content, ALT and AST activities did not differ between all treatments (P > 0.05). Serum MDA levels in the HL group were higher than those in the CON, HLG, and HLA groups, although the differences were not statistically significant (P > 0.05).

Fig. 1.

Fig. 1

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Effect of GSH and AX supplement in high-lipid diets on serum parameters of Litopenaeus vannamei. Data are expressed as means ± SE (n = 3). Different lowercase letters indicate significant differences (P < 0.05).

3.4. Intestine histology

Representative images of the intestinal histomorphology for all treatments were shown in Fig. 2. Shrimp fed the HLG diet demonstrated superior intestinal health, with significantly greater IWT and VH compared to the HL and HLA groups (P < 0.05; Table 2). The IWT in the HLG group was statistically equivalent to that of the CON group (P < 0.05). Furthermore, the VW did not differ significantly among the CON, HL, and HLA groups (P > 0.05); however, VW in these groups was markedly lower than in group HLG (P < 0.05).

Fig. 2.

Fig. 2

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Effect of AX and GSH supplement in high-lipid diets on intestinal histology of Litopenaeus vannamei. All sections of intestine tissue were stained with H&E. Scale bar 50 μm, VH: Villus height, VW: Villus width, IWT: Intestinal wall thickness.

Table 2.

Effect of dietary AX and GSH supplement in high-lipid diets on intestinal morphology parameters of shrimp.

Index
 Diets
CON HL HLG HLA
IWT (μm) 92.88 ± 4.74b 51.71 ± 5.97a 87.06 ± 4.84b 64.07 ± 3.78a
VH (μm) 44.16 ± 3.28b 33.14 ± 1.40a 43.49 ± 2.09b 33.86 ± 0.92a
VW (μm) 29.08 ± 1.92a 24.24 ± 1.90a 39.36 ± 0.70b 24.74 ± 2.30a
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Data are expressed as means ± SE (n = 3). Different lowercase letters indicate significant differences (P < 0.05).

3.5. Hepatopancreas histology

It can be seen from Fig. S1 that hepatopancreas tubules of all treatments were observed to be close and well aligned with each other. The lumen stellate polygonal structure was regular and clear, and none of them had obvious tissue damage. Distinctive cellular compositions were observed. The HLG group exhibited a higher prevalence of R-cells, while the HL and HLA groups were characterized by an abundance of B-cells.

3.6. Lipid metabolism-related gene expression and TG content in hepatopancreas

The HL group exhibited significantly elevated hepatopancreatic TG content in contrast with all other groups (P < 0.05; Fig. 3). TG content in group HLA did not differ significantly from that in group HLG (P > 0.05), but was remarkably higher in contrast with group CON (P < 0.05). The expression of fas gene did not differ significantly between the HLG and HLA groups (P > 0.05), and both were notably lower than in groups HL and CON (P < 0.05). No significant differences in the gene expression levels (srebp, ampk, cpt1, fabp, and fatp) between all treatments (P > 0.05). The HLG group showed significantly higher g6pdh expression in contrast with the HLA group (P < 0.05). Compared to the CON group, lipid catabolism-related genes (tgl, acox, and pmfe) were notably higher in the HL, HLG, and HLA groups (P < 0.05). Additionally, the HLA group exhibited significantly lower tgl expression compared with the HLG group (P < 0.05).

Fig. 3.

Fig. 3

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Effect of GSH and AX supplement in high-lipid diets on triglyceride content and lipid metabolism-related gene expression in hepatopancreas of Litopenaeus vannamei. AMP-activated protein kinase (ampk), glucose-6-phosphatedehydrogenase (g6pdh), fatty acid synthase (fas), sterol-regulatory element binding protein (srebp), fatty acid transport protein (fatp), carnitine O-palmitoyltransferase 1 (cpt1), fatty acid binding protein (fabp), triacylglycerol lipase (tgl), peroxisomal multifunctional enzyme type 2 (pmfe), peroxisomal acyl-coenzyme A oxidase (acox). Data are expressed as means ± SE (n = 3). Different lowercase letters indicate significant differences (P < 0.05).

3.7. Antioxidant and immune-related enzymes in hepatopancreas

Analysis of hepatopancreatic antioxidant status revealed distinct effects of the dietary supplements (Fig. 4). While MDA content and PO activity remained unchanged across all groups, both the HLG and HLA groups exhibited a significantly enhanced T-AOC compared to the CON group (P < 0.05). The HLG group demonstrated significantly higher SOD activity than the HLA group (P < 0.05). Furthermore, the HLG group showed markedly higher activity of GSH-Px and CAT and GSH content than the CON group (P < 0.05). The GSH content was no significant difference observed between groups HLG and HLA (P > 0.05), while remarkably higher than that in group HL (P < 0.05).

Fig. 4.

Fig. 4

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Effect of GSH and AX supplement in high-lipid diets on antioxidant enzyme activities in hepatopancreas of Litopenaeus vannamei. Data are expressed as means ± SE (n = 3). Different lowercase letters indicate significant differences (P < 0.05).

ACP activity in the HLG group showed no significant difference from the HL and HLA groups but a significant increase compared to the CON group (P < 0.05; Fig. 5). AKP activity was significantly elevated in both the HLG and HLA groups compared to the HL and CON groups (P < 0.05).

Fig. 5.

Fig. 5

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Effect of GSH and AX supplement in high-lipid diets on immune-related enzyme activities in hepatopancreas of Litopenaeus vannamei. Data are expressed as means ± SE (n = 3). Different lowercase letters indicate significant differences (P < 0.05).

4. Discussion

The optimal lipid requirements of Litopenaeus vannamei are influenced by factors such as species, culture stages, genetic strains, diet composition, and rearing practices (El-Sayed, 2021; Emerenciano et al., 2022). Likewise, Litopenaeus vannamei exhibits intolerance to high-lipid diets (Chuphal et al., 2021; Xu et al., 2018), which is attributed to its inefficient lipid emulsification system. This limited capacity of shrimp, when confronted with high-lipid diets, imposes oxidative stress (Xu et al., 2018) and metabolic disorders (Liu et al., 2022a), forcing energy allocation to shift from growth toward maintaining homeostasis and countering stress responses, ultimately suppressing growth indicators. The negative effects of high-lipid diets were demonstrated in this study, as shrimp fed the HL diet exhibited significantly reduced growth performance (FBW, WGR, and SGR) compared to those fed the CON diet. Supplementing AX in high-lipid diets slightly improves shrimp growth, though not significantly. However, adding GSH to high-lipid diets substantially mitigates the negative growth effects of high-lipid diets, restoring shrimp growth performance to CON levels.

A well-developed intestinal morphology is crucial for enhancing the digestive capacity and promoting growth in Litopenaeus vannamei (Won et al., 2020; Wu et al., 2022). The present study demonstrated that a high-lipid diet (HL group) significantly impaired intestinal development in shrimp, as evidenced by a markedly lower IWT and VH compared to the CON group. The reason may be that the high-lipid diet induced oxidative stress (Raghuvaran et al., 2023) and inflammation (Dai et al., 2022), which in turn affected the intestinal development. GSH improved intestinal morphology in shrimp (Wang et al., 2018c), which was reflected in the HLG group with higher IWT, VW and VH than both the HL and HLA groups, as well as comparable apparent phenomena of shrimp in the CON group. These results indicated that dietary supplementation with GSH effectively amelioratesd intestinal morphology in Litopenaeus vannamei fed a high-lipid diet.

The hepatopancreas is the primary site of nutrient storage, metabolism, secretion, and detoxification in crustaceans (Liang et al., 2022). Dietary composition may affect the structure of the hepatopancreas, while nutritional imbalances may alter its morphology and function (Jahromi et al., 2021). When hepatopancreas exhibited a decrease in B-cells accompanied by an increase in R-cells, a remarkable increase in shrimp growth was observed (Abidin et al., 2022; Romano et al., 2015). R-cells, involved in nutrient uptake, metabolism, mineral excretion, and lipid storage (Vogt, 2019), its count related to the healthier hepatopancreas and facilitated hepatopancreas energy reserves (Pourmozaffar et al., 2019; Zhao et al., 2018), which promoted rapid growth in rock lobster (Jasus edwardsii) (Johnston et al., 2003). B-cells play a central role in crustaceans by specializing in nutrient digestion, as well as the synthesis and secretion of key digestive enzymes (Abidin et al., 2022). In this study, shrimp hepatopancreas in the HLG group displayed with more R-cells and fewer B-cells than the other groups, alongside significantly improved growth performance. The vesicle area and proportion of B-cells in hepatopancreas were significantly higher in the HL and HLA groups than in the HLG and CON groups. High-lipid diets may contribute to excessive lipid accumulation in hepatopancreas, necessitating increased digestive enzyme activity for efficient processing. It would be a compensatory manifestation within the shrimp's body. However, GSH may reduce lipid accumulation by regulating lipid metabolism gene expression (Chen et al., 2025), thereby showing a better hepatopancreas structure.

Lipid metabolism plays a pivotal role in numerous physiological and biochemical functions across living organisms. Higher dietary lipids can lead to nutritional imbalances and increased metabolic stress, potentially altering key metabolic pathways (Wellen & Thompson, 2010) and associated with lipid accumulation, especially in the hepatopancreas (Dai et al., 2022; Liu et al., 2022b; Naiel et al., 2023).

As the central enzyme of the endogenous lipogenesis pathway, fatty acid synthase (FAS) is directly involved in regulating lipid deposition, making it a critical marker for assessing dietary effects (Schroeder et al., 2021). The hepatopancreas of shrimp in the group HL displayed a notable elevation in TG content compared to group CON, whereas the expression of the fas gene was markedly downregulated. These findings aligned with previous studies on greasyback shrimp (Metapenaeus ensis) (Chen et al., 2024), where high-lipid diets similarly suppressed fas expression and increased TG contents. It should be noted that TG contents in the hepatopancreas were not solely governed by endogenous synthesis, but were more influenced by the balance between fat input and output in hepatopancreas. But high-lipid diets disrupted this balance. The suppression of fas gene may be attributed to elevated levels of dietary polyunsaturated fatty acids or lipids, which inhibit endogenous lipid synthesis. Increased dietary lipid intake reduced the need for endogenous lipid production, likely contributing to the downregulation of fas expression (Leng et al., 2012). However, other studies have reported upregulation of gene fas in shrimp responsed to high-lipid diets, potentially attributed to differences in dietary lipid sources (Dai et al., 2022; Peng et al., 2017). Interestingly, the addition of GSH or AX to high-lipid diets resulted in lower fas expression and reduced hepatopancreas TG content compared to the HL group. It suggested that GSH and AX supplementation suppress lipid synthesis by downregulating fas expression, thereby alleviating lipid deposition caused by high-lipid diets. Similar findings have been reported in mice, where AX inhibited the increase in liver TG levels induced by high-lipid diets (Ikeuchi et al., 2007). Additionally, g6pdh gene expression was notably up-regulated in hepatopancreas of group HLG in contrast with group HL. As the key enzyme of the pentose phosphate pathway, G6PDH generates NADPH, which is essential for fatty-acid and steroid biosynthesis (Zhou et al., 2020). This upregulation implies that GSH may enhance lipid absorption and utilization by influencing the pentose phosphate pathway.

Lipid catabolism-related genes, such as triacylglycerol lipase (tgl), peroxisomal acyl coenzyme A oxidase (acox), and peroxide multifunctional type 2 (pmfe), were remarkably upregulated in hepatopancreas of the HL group in contrast with the CON group. It indicated that high-lipid diets promote fatty acid β-oxidation and lipolysis, likely as a self-protective mechanism to counteract excessive lipid accumulation. However, this response appears insufficient to fully mitigate hepatopancreatic lipid deposition, as observed in zebrafish (Dai et al., 2015) and trout (Oncorhynchus mykiss) (Zhao et al., 2023) fed high-lipid diets. Interestingly, the expression of tgl gene in the HLA group was significantly down-regulated compared to that in the HL group, possibly due to the reduced TG content in hepatopancreas of the HLA group, which may help maintain lipid homeostasis. These findings suggested that high-lipid diets significantly affect lipid metabolism in shrimp.

In response to excessive dietary lipids, shrimp up-regulate fatty acid β-oxidation and down-regulate lipid synthesis-related genes, such as fas, to reduce endogenous lipid production. However, the capacity of shrimp to catabolize lipids may be exceeded, leading to lipid accumulation and metabolic stress. Supplementation with GSH or AX in high-lipid diets effectively reduced TG content in the hepatopancreas by inhibiting endogenous lipid synthesis, thereby alleviating lipid deposition. These additives may modulate lipid metabolism pathways, protect the hepatopancreas, and help shrimp cope with the challenges of nutritional stress induced by high-lipid diets. GSH acts primarily as a lipid metabolism promoter and is the strong upregulation of the tgl gene, enhancing the catabolism of stored triglycerides. Supplementing with GSH or AX in high-lipid diets can suppress endogenous lipid synthesis by downregulating the fas gene expression levels, effectively reducing triglyceride levels in hepatopancreas, thereby alleviating lipid deposition.

β-oxidation is the major pathway responsible for breaking down fatty acids to meet cellular energy demands (Houten et al., 2016; Houten & Wanders, 2010), which is also the main metabolism pathway for fatty acid degradation in shrimp (Lu et al., 2023). High-lipid diets often result in excessive lipid accumulation, promoting oxidative stress (Xie et al., 2020) and associate with the excessive generation of reactive oxygen species (ROS). Overabundance of ROS has the potential to compromise cellular integrity and functionality (Schieber & Chandel, 2014), stimulate the transcription of the pro-inflammatory cytokine TNF-α by activating NF-κB (Morita et al., 2013). And then it lead to body inflammation (Mittal et al., 2014), cellular damage and death (Dixon & Stockwell, 2014). Organisms rely on antioxidant systems to neutralize ROS (Loboda et al., 2016). Consequently, the antioxidant defense system is critical for protecting crustaceans from ROS-induced damage and is influenced by factors such as diet, environment, and health conditions (Frías-Espericueta et al., 2022). Dietary GSH supplementation has been reported to increase CAT and GSH-Px activities in Litopenaeus vannamei (Wang et al., 2018a; Xia & Wu, 2018) and Chinese mitten crab (Eriocheir sinensis) (Liu et al., 2020c). Similarly, dietary AX supplementation has been shown to enhance CAT and GSH-Px activities in Litopenaeus vannamei (Eldessouki et al., 2022; Wang et al., 2020). This study demonstrates that shrimp fed the HL diet showed a non-significant rise in GSH-Px and T-AOC alongside an increase in MDA—indicating a compensatory response to oxidative stress. Both GSH and AX effectively enhanced the antioxidant enzyme system (GSH-Px and CAT) of shrimp challenged by a high-lipid diet. Notably, SOD activity in the HLG group was elevated relative to the HLA group, which indicated that GSH and AX, although both antioxidants, exerted different functions. Consistent with previous findings (Sun et al., 2025; Xia & Wu, 2018), GSH can enhance SOD activity in shrimp, whereas AX does not exhibit this effect. Although the differences did not reach statistical significance, CAT and GSH-Px activities were also elevated in the HLG group relative to the HLA group. These findings suggest that GSH may be more effective than AX in enhancing activities of key antioxidant enzymes, including GSH-Px, CAT, and SOD. These findings indicated that while both additives were beneficial, GSH provides a superior capacity to upregulate critical antioxidant enzymes.

The non-enzymatic antioxidant system in Litopenaeus vannamei includes small molecule compounds such as ascorbic acid, AX, and GSH, which play crucial roles in ROS scavenging (Frías-Espericueta et al., 2022). GSH is the primary endogenous low-molecular-weight antioxidant, characterized by its reactive thiol groups, which can directly react with ROS to reduce the oxidative damage to the cells or act as a substrate for glutathione peroxidase to be oxidized to GSSH, performing its antioxidant function (Liu et al., 2022a; Ribas et al., 2014; Srikanth et al., 2013).

GSH has been shown to reduce ROS in the liver of grass carp (Ctenopharyngodon idella) (Ming et al., 2019) and common carp (Cyprinus carpio) (Xue et al., 2022), as well as in hepatopancreas of Chinese mitten crab (Eriocheir sinensis) under lipopolysaccharide-induced oxidative stress (Liu et al., 2020b). AX is a non-enzymatic and potent antioxidant that prevents chain reactions by bursting unilinear oxygen species and scavenging free radicals (Brotosudarmo et al., 2020), reducing the production of hemolymph ROS by Chinese mitten crabs (Eriocheir sinensis) under high pH stress (Wang et al., 2018a), inhibiting excessive hemocyte ROS production induced by high temperature stress in pufferfish (Takifugu obscurus) (Cheng et al., 2018), and alleviating the oxidative stress damage induced by oxidized fish-oil diet in Litopenaeus vannamei (Yu et al., 2020). In contrast with the CON group, hepatopancreas GSH content was lower in group HL, although there was no statistically significant difference, which may be attributed to the increased production of ROS induced by high-lipid diets depleting the antioxidant GSH (Qu et al., 2022). Shrimp of the HLG group exhibited significantly elevated hepatopancreas GSH content compared with the HL group, suggesting that shrimp fed the HLG diets increased GSH levels, alleviating the depletion caused by high-lipid diets and stabilizing the glutathione system. A similar result with GSH supplementation to increase hepatopancreas GSH content of Chinese mitten crab (Eriocheir sinensis) (Liu et al., 2020c).

In contrast, the shrimp fed the HLA diet maintained GSH content at a level comparable to the CON group, not by increasing its concentration, but likely through its potent intrinsic ability to directly scavenge ROS, thereby reducing the metabolic burden on the glutathione system. This mechanistic distinction is critical for evaluating their efficacy. While both additives achieved a similar T-AOC, GSH (HLG group) demonstrated a superior ability to support and enhance the endogenous glutathione system directly. Though less effective at elevating specific antioxidant enzymes, AX (HLA group) contributed equally to the antioxidant capacity through its strong direct free-radical scavenging activity. Therefore, GSH (HLG group) acted more as a synergistic regulator of the endogenous antioxidant system, whereas AX (HLA group) showed functions as a powerful exogenous scavenger, with both strategies being effective in mitigating oxidative stress.

The nonspecific immune system serves as the primary defense mechanism in crustaceans against invasive pathogens (Huang & Ren, 2020). Enzymes such as ACP and AKP contribute to pathogen recognition and phagocytosis by modifying their surface structure (Liu et al., 2020a; Zhu et al., 2022). In this study, compared with groups HL and HLA, the HLG group exhibited increased hepatopancreas ACP activity, although the difference was not statistically significant, implying a potential beneficial effect of GSH supplementation in high-lipid diets on ACP activity. Dietary GSH could boost AKP activity in Litopenaeus vannamei (Xia & Wu, 2018) and Chinese mitten crab (Eriocheir sinensis) (Liu et al., 2020c). Similarly, dietary AX supplementation has been shown to increase AKP activity in Litopenaeus vannamei (Liu et al., 2022b). Consistent with these reports, the present study found that dietary inclusion of either GSH or AX notably increased AKP activity in hepatopancreas of shrimp fed high-lipid diets. These results indicated that both supplements could enhance the ability of shrimp to withstand pathogenic challenges through increasing AKP activity.

5. Conclusion

Overall, either GSH or AX supplementation in high-lipid diets could reduce hepatopancreatic lipid accumulation and effectively alleviate oxidative stress in Litopenaeus vannamei. However, their mechanisms of action differed, leading to distinct practical advantages. GSH demonstrated superior efficacy in directly regulating lipid metabolism, reinforcing the endogenous antioxidant enzyme system, and improving intestinal health to enhance growth performance.

CRediT authorship contribution statement

Jiandong Chen: Writing – original draft, Validation, Methodology, Investigation, Formal analysis, Conceptualization. Tao Cheng: Validation, Methodology, Investigation, Formal analysis. Xiang Si: Validation, Methodology, Investigation, Formal analysis. Fan Yang: Resources, Methodology, Investigation. Jianhua Yi: Resources, Methodology, Investigation. Zhilong Yang: Resources, Methodology, Investigation. Zhaowen Li: Resources, Methodology, Investigation. Beiping Tan: Investigation, Funding acquisition, Conceptualization. Chunfeng Yao: Supervision, Methodology, Investigation, Conceptualization. Shuyan Chi: Writing – review & editing, Project administration, Conceptualization.

Funding

This work was supported financially by the National Key Research and Development Program of China (2023YFD2402000).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.fochms.2025.100331.

Contributor Information

Chunfeng Yao, Email: Xiaoyao3221@163.com.

Shuyan Chi, Email: chishuyan77@163.com.

Appendix A. Supplementary data

Supplementary material

mmc1.docx (373KB, docx)

Data availability

Data will be made available on request.

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