Organic manganese as a functional micronutrient: Promoting metabolic health and sustainable growth in Litopenaeus vannamei

Abstract

This study evaluated the effects of five manganese (Mn) sources on Pacific white shrimp (Litopenaeus vannamei). Five Mn sources include Mn sulfate (MnSO₄·H₂O), Mn dioxide (MnO₂), Mn dioxide nanoparticles (MnO₂NPs), Mn glycine chelate (Mn-Gly), and Mn chelate of hydroxy analogue of methionine (Mn-MHA). During a 56-d feeding trial, 450 shrimp (1.03 ± 0.02 g) were randomly distributed among five dietary treatments, with each having three replicates. No significant differences in growth performance and feed utilisation were found among different Mn sources (P > 0.05). However, organic Mn sources (Mn−Gly and Mn−MHA) significantly enhanced Mn deposition (P = 0.002) and upregulated a key Mn transporter (zip14, tmem165, and fpn1) in the hepatopancreas when compared with inorganic MnO₂ source (P < 0.05). Moreover, dietary organic Mn-MHA significantly enhanced systemic antioxidant capacity (total-antioxidant capacity [T-AOC] and glutathione peroxidase [GSH-Px]) and innate immune response, as evidenced by increased activities of relevant enzymes (acid phosphatase [AKP], acid phosphatase [ACP], and nitric oxide synthase [NOS]) and upregulation of associated genes (gpx, cat, sod, nos, and akp) when compared with MnO₂ (P < 0.05). Furthermore, the organic Mn sources significantly downregulated genes related to endoplasmic reticulum stress (perk, atf4, atf6, and eif2α), inflammation (spz-5, il-16, and rab6a), and apoptosis (caspase8, p53, and caspase3), while significantly upregulating the anti-apoptotic gene (bl-1) when compared with MnO₂ (P < 0.05). Notably, Mn−MHA supplementation significantly reduced hepatopancreatic lipid content (P = 0.002) by modulating the expression of genes involved in lipid synthesis (fas and acc) and lipolysis (aco and cpt) (P < 0.05). In summary, although growth remained unaffected, organic Mn sources, particularly Mn−Gly and Mn−MHA, demonstrated superior bioavailability by enhancing antioxidant status, immune function, and hepatopancreas health as well as by regulating lipid metabolism.

1. Introduction

Manganese (Mn) is an essential trace mineral for aquatic animals; its bioavailability from dietary sources highly influences a range of physiological processes central to growth, development, and health (Lall and Kaushik, 2021; Sliva et al., 2019). The effectiveness of Mn in being a cofactor for the enzymes involved in fatty acid and amino acid metabolism as well as its role in enhancing the antioxidant activity of SOD (The full names of the abbreviations of all genes and proteins can be found in Table S1) and its involvement in muscle and bone metabolism are fundamentally dependent on its absorption and utilization efficiency (Andreini et al., 2008; Aschner and Ashner, 2005; Huang et al., 2025). Similarly, Mn regulates lipid and energy metabolism and thereby enhances lipid utilisation (Xiang et al., 2022). Establishing the dietary Mn requirement is crucial, with reported values ranging from 2.50 to 25.00 mg/kg for fish and 23.90 to 32.06 mg/kg for shrimp (Cai et al., 2017; Lall and Kaushik, 2021). Notably, the manifestation of Mn deficiency pathologies, such as chondrocyte damage in the tibial growth plate (Wang et al., 2021) as well as growth inhibition and intestinal inflammation in grass carp (Cyprinus carpio) (Jiang et al., 2017; Tang et al., 2016), highlights the need to ensuring adequate intake, and high bioavailability of Mn in the diet. As seawater provides insufficient Mn (0.01 mg/L) for marine crustaceans, dietary supplementation becomes necessary (Jiao et al., 2021; Li et al., 2025; NRC, 2011), emphasising the importance of selecting the optimal Mn source.

Although meeting the quantitative requirement is fundamental, the chemical form of dietary Mn is a key determinant of its bioavailability and subsequent physiological efficacy. Feed Mn sources of aquatic animals are mainly classified into inorganic salts (e.g. Mn sulfate [MnSO₄·H₂O], Mn dioxide [MnO₂], and Mn dioxide nanoparticles [MnO₂NPs]) and organic complexes (e.g. Mn glycine chelate [Mn-Gly] and Mn chelate of hydroxy analogue of methionine [Mn-MHA]) (Nguyen et al., 2019; Sliva et al., 2019; Yin et al., 2024). A growing body of evidences suggests that organic Mn sources often demonstrate superior bioavailability by preventing the strong absorption of a trace element into insoluble colloids compared with their inorganic counterparts (Buentello et al., 2009; Lin et al., 2013; Nguyen et al., 2019). This advantage is attributed to their unique chemical characteristics (Fly et al., 1989; Henry et al., 1989). It is believed that organic Mn, such as amino acid chelates, is absorbed via short peptides and/or hydrolysed proteins with coordinate covalent bonds, which further prevents antagonism from phytic acid and avoids the interaction of Mn²⁺ with anti-nutritional factors and other minerals, resulting in efficient intestinal Mn absorption and tissue retention (Byrne and Murphy, 2022; Michalke and Fernsebner, 2014; Truong et al., 2022). For example, Nie et al. (2016) reported higher bioavailability for organic Mn in juvenile cobia (Rachycentron canadum L). Recently, dietary Mn-MHA and Mn-Gly enhanced the growth performance and intestinal health of yellow catfish (Pelteobagrus fulvidraco) (Xu et al., 2023), highlighting the functional benefits of organic Mn beyond mere absorption. However, despite these promising findings in fish and terrestrial livestock, a systematic evaluation of a broad spectrum of Mn sources is absent in crustaceans, and their comparative impacts on pivotal physiological processes and bioavailability remain largely unknown. As a central metabolic organ (Luo et al., 2021) and a key regulator of Mn homeostasis (Liu et al., 2021) in crustaceans, the hepatopancreas is highly susceptible to dietary and environmental stresses. Therefore, this study aims to address these issues by comprehensively comparing five Mn sources and investigating their specific effects on Litopenaeus vannamei, with a particular focus on hepatopancreatic health.

L. vannamei is a globally important aquaculture species; its production in China has reached 216,478 tons in 2024 (MARA, 2025; Sangklai et al., 2025). The previous study determined that the optimal dietary Mn requirement for L. vannamei should be 30.90 mg/kg (Li et al., 2025). However, simply fulfilling the quantitative requirement is insufficient for optimising physiological resilience and overall health under intensive farming conditions. Although inorganic Mn sources can satisfy the basic growth requirement, it is hypothesized that organic Mn forms offer superior benefits owing to their higher bioavailability, which can translate into enhanced antioxidant capacity, improved immune function and better tissue health—outcomes that can justify their use despite the high initial cost of organic Mn sources. Therefore, this study aims to comprehensively explore and compare the effects of five dietary Mn sources (MnSO₄·H₂O, MnO₂, MnO₂NPs, Mn-Gly, and Mn-MHA) on growth, hepatopancreas health, lipid metabolism, and endoplasmic reticulum stress (ERS) to provide a scientific basis for cost-effective and sustainable Mn source selection in L. vannamei feeds.

2. Materials and methods

2.1. Animal ethics statement

All shrimp-related procedures were approved by the Animal Care and Use Committee of Ningbo University (approval No. SYXK20190005) and complied by the Regulation on the Administration of Laboratory Animals in Zhejiang Province (Zhejiang Provincial Government Order No. 263, released on August 17, 2009).

2.2. Diet formulation, experimental design, and sample collection

Five isonitrogenous and isolipidic diets were formulated via supplementation with different Mn sources (MnSO₄·H₂O, MnO₂, MnO₂NPs, Mn-Gly, and Mn-MHA) to meet nutritional requirements (Table 1). MnSO₄·H₂O (99% purity) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). MnO₂ (99% purity) and MnO₂NPs (99.5% purity) were purchased from Shandong Xiya Chemical Co., Ltd. (Linyi, Shandong, China). Mn-Gly (99% purity) and Mn-MHA (≥95% purity) were sourced from Hebei Hontao Bioengineering Co., Ltd. (Shijiazhuang, Hebei, China) and Changsha Xingjia Bioengineering Co., Ltd. (Changsha, Hunan, China), respectively. The five Mn sources were supplemented to the basal diet based on the elemental Mn content to achieve a comparable target total Mn concentration of about 31.00 mg/kg diet. The basal diet was analysed to contain 16.30 mg/kg Mn from intrinsic ingredients. The supplemental Mn was calculated, assuming the following Mn contents for each source: MnSO₄·H₂O (32%), MnO₂ (63%), MnO₂NPs (63%), Mn-Gly (20%), and Mn-MHA (12%). Accordingly, the respective compounds were added at the following levels (mg/kg diet): 44.75, 23.02, 22.90, 72.00, and 120.00 mg/kg diet, respectively. The actual dietary Mn contents were determined via inductively coupled plasma atomic optical emission spectrometry (ICP-OES) were 31.94, 30.79, 30.69, 30.97, and 30.53 mg/kg (average 30.98 mg/kg). Briefly, all macro-ingredients, such as Peru fishmeal, soybean meal, peanut meal, poultry by-product meal, hemocyte albumen powder, and shrimp shell meal, were finely ground, passed through a 60-mesh screen and accurately weighed. After mixing of the macro-ingredients, all micro-components, such as Mn-free vitamin and mineral premixes, Ca(H₂PO₄)₂, sodium humate, and the respective Mn source, were accurately weighed and mixed with macro-ingredients using a stepwise expansion method to make a mix. Subsequently, soybean lecithin and fish dissolved pulp were added to the feed mixture. After thorough mixing, water was added and both were blended together in the mixer to obtain a homogenous mixture. Pellets of 1.5 and 2.0 mm diameter were produced using a pellet feed maker (F-26, South China University of Technology Machine Factory, Guangzhou, Guangdong, China). Finally, the pellets were oven-dried at 60 °C and stored at −20 °C.

Table 1.

Ingredients and proximate composition of experimental diets (%, dry matter basis).

Items	Dietary Mn sources3
	MnSO4·H2O	MnO2	MnO2NPs	Mn-Gly	Mn-MHA
Ingredients
Peru fish meal	30.00	30.00	30.00	30.00	30.00
Soybean meal	18.00	18.00	18.00	18.00	18.00
Peanut meal	6.00	6.00	6.00	6.00	6.00
Poultry by-product meal	5.00	5.00	5.00	5.00	5.00
Fish dissolved pulp	7.00	7.00	7.00	7.00	7.00
Hemocyte albumen powder	2.50	2.50	2.50	2.50	2.50
Shrimp shell meal	5.00	5.00	5.00	5.00	5.00
Wheat flour	20.80	20.80	20.80	20.80	20.80
Soybean lecithin	2.00	2.00	2.00	2.00	2.00
Vitamin premix1	0.30	0.30	0.30	0.30	0.30
Mineral premix2	1.00	1.00	1.00	1.00	1.00
Sodium humate	0.40	0.40	0.40	0.40	0.40
Ca(H2PO4)2	2.00	2.00	2.00	2.00	2.00
Total	100.00	100.00	100.00	100.00	100.00
MnSO4·H2O, mg/kg	44.75	0.00	0.00	0.00	0.00
MnO2, mg/kg	0.00	23.02	0.00	0.00	0.00
MnO2NPs, mg/kg	0.00	0.00	22.90	0.00	0.00
Mn-Gly, mg/kg	0.00	0.00	0.00	72.00	0.00
Mn-MHA, mg/kg	0.00	0.00	0.00	0.00	120.00
Proximate composition
Dry matter	88.44	89.17	88.44	88.38	88.57
Crude protein	44.95	44.26	45.08	44.16	44.65
Crude lipid	11.26	10.21	10.40	10.03	10.18
Organic matter	77.57	78.54	77.60	77.02	77.74
Mn, mg/kg	31.94	30.79	30.69	30.97	30.53

¹Vitamin premix (g/kg): retinyl acetate, 1.2121; cholecalciferol, 1.2000; all-rac-α-tocopherol, 20.0000; menadione, 9.0909; thiamine, 10.8696; riboflavin, 7.5000; ascorbic acid, 30.0000; pyridoxine hydrochloride, 12.1212; cyanocobalamin, 2.0000; folic acid, 40.0000; biotin, 12.5000; nicotinic acid, 40.4040; D-Ca pantothenate, 16.1290; inositol, 204.0816; cellulose, 592.8916.

²Mineral premix (g/kg): CuSO₄·5H₂O (99%), 15.87; KIO₃ (99.8%), 0.02; MgSO₄·7H₂O (99%), 358.45; K₂SO₄ (99%), 225.09; NaCl (99.5%), 76.64; C₆H₁₀O₆Ca·5H₂O (98%), 51.13; CoSO₄·7H₂O (99.5%), 2.40; ZnSO₄·7H₂O (99.5%), 26.52; zeolite, 243.88. The mineral premix is not supplemented with Mn.

³MnSO₄·H₂O, Mn sulfate; MnO₂, Mn dioxide; MnO₂NPs, Mn dioxide nanoparticles; Mn-Gly, Mn glycine chelate; and Mn-MHA, Mn chelate of hydroxy analogue of methionine.

A 56-d feeding trial was conducted following a 2-week acclimation. At the Ningbo Marine Fisheries Science and Technology Innovation Base (Ningbo, Zhejiang, China), 450 shrimp (1.03 ± 0.02 g) were randomly assigned to five treatment groups, with three replicates/group and 30 shrimp/replicate, housed in 300-L tanks (effective volume: 250 L). The shrimp were hand-fed their respective diets three times daily (06:30, 11:00, and 18:00). The daily ration was set at 6%–8% of their body weight (BW) in each tank and was adjusted every 2 weeks. During each feeding event, feed was gradually provided until apparent satiation was reached, which was determined via cessation of active feeding behaviour. Water parameters were maintained as follows: seawater temperature 27.7 ± 2.0 °C, salinity 23.30−25.60 practical salinity unit (PSU), pH 7.70−8.30, and dissolved oxygen > 6 mg/L. The shrimp were weighed every 2 weeks, and the feeding rates were adjusted accordingly. Pellet size was adjusted based on the shrimp size.

After the 8-week feeding trial, all surviving shrimp in each replicate tank were counted and collectively weighed to determine the total biomass. The final average BW was calculated by dividing the total final biomass by the number of surviving shrimp in that replicate. Furthermore, growth performance indices, including percent weight gain (PWG) and specific growth rate (SGR), were calculated for each replicate based on the initial and final average weights derived from the total biomass. Each replicate was evaluated for feed efficiency and survival rate based on daily records of feed input and mortality. Before sampling, shrimp were anesthetised on ice to minimise suffering. Subsequently, four shrimp/replicate were randomly selected to measure individual body length and weight. Their hepatopancreas were dissected and weighed to calculate the hepatosomatic index and condition factor. Hemolymph was collected from the ventral sinus (pooled samples from five shrimp/replicate, n = 3 biological replicates/treatment), centrifuged (3000 × g, 10 min, 4 °C) to obtain the supernatant and then stored at −80 °C. Hepatopancreatic samples (pooled samples from three shrimp/replicate, n = 3 biological replicates/treatment) were dissected, immediately frozen in liquid nitrogen and then stored at −80 °C in a tube (containing RNA-later solution) for gene expression analysis. Additional intestine, hepatopancreas, and muscle samples (pooled samples from three shrimp/replicate, n = 3 biological replicates/treatment) were frozen and stored at −80 °C for enzymatic and composition analyses.

2.3. Determination of Mn contents and proximate composition

The Mn contents in diets and tissues (muscle, hepatopancreas, and carapace) were determined via ICP-OES (Varian Medical Systems, Inc., Palo Alto, CA, USA) following a wet digestion process with the GB 5009.268-2025 (China National Standard, 2025). Briefly, all samples were initially pulverised using a freeze-drying technology. Approximately 0.5 g (accurate to 0.001 g) of each homogenised sample was digested with 10 mL of a nitric-perchloric acid mixture on an electric hotplate (150 °C, 5 h) until the solution became clear and colourless. After cooling, the digest was diluted to a final volume of 50 mL for analysis. A multi-point external curve was created using certified Mn standard solutions (0.000, 0.005, 0.050, 0.200, 0.500, and 1.000 mg/L), which demonstrated excellent linearity with a correlation coefficient (R²) > 0.9999. The instrumental operating conditions were set as follows: axial view for observation, RF power of 1200 W, plasma gas flow rate of 15 L/min, nebuliser gas flow rate of 0.65 L/min, and an analytical pump speed of 50 r/min. The limits of detection and quantification for Mn were found to be 0.1 and 0.3 mg/kg, respectively. To ensure analytical accuracy, a single-element Mn standard stock solution was processed with each sample batch. The test report indicated that for samples with a Mn content > 1.000 mg/kg, the absolute difference between two independent results obtained under repeatability conditions should not exceed 10% of the arithmetic mean. The accuracy and reliability of the analytical results were confirmed via quality control using certified reference materials (CRMs) with low (infant cereal CRM; GBW10279) and high (rice flour CRM; TMQC1495) Mn contents.

The chemical compositions of diets, tissues (muscle and hepatopancreas), and whole shrimp were analysed following the AOAC (1995). Dry matter was gravimetrically analysed via oven-drying by an Electric thermostatic drying oven (DHG-9240 A, Shanghai Yiheng Scientific Instrument Co., Ltd., Shanghai, China) at 105 °C to a constant mass (method 934.01). Crude lipid used an extraction analyser (SX360, OPSIS AB, Furulund, Skåne, Swden) based on the Soxhlet extraction method (method 920.39). Crude protein content used the Dumas combustion method (method 990.03) by a Dumas Nitrogen Analyser (FP-528, Leco Corporation, San Jose, CA, USA). And ash was via combustion in a muffle furnace (TM-2010, Beijing Innochem Science & Technology Co., Ltd., Beijing, China) at 550 °C for 8 h (method 924.05). Organic matter of diets was calculated as dry matter minus crude ash (Hasanthi et al., 2025).

2.4. Biochemical indices of the hemolymph and tissues

Approximately 1.0 g hepatopancreas and intestine samples were homogenised with a 9-fold volume of ice-cold sterile saline using a mechanical homogeniser (T25 digital ULTRA-TURRAX, IKA-Werke GmbH & Co. KG, Staufen, Breisgau, Germany) on an ice bath. Subsequently, the homogenates were centrifuged (4000 × g, 4 °C, 10 min) to obtain the supernatants for biochemical analysis. The levels of triglyceride (TG; A110-1-1), total-cholesterol (T-CHO; A111-1-1), and malondialdehyde (MDA; A003-1-2) as well as activities of alkaline phosphatase (AKP; A059-2-2), nitric oxide synthase (NOS; A014-2-2), total-antioxidant capacity (T-AOC; A015-1-2), acid phosphatase (ACP; A060-2-1), and glutathione peroxidase (GSH-Px; A005-1-1) in the hepatopancreas were determined using commercial kits (Nanjing Jiancheng Bioengineering Institute Co., Ltd., Nanjing, Jiangsu, China) and a microplate reader (SpectraMax M2, Molecular Devices, Silicon Valle, CA, USA) or an ultraviolet spectrophotometer (UV-1800, SHIMADZU Corporation, Kyoto, Japan). All assays were strictly performed according to the manufacturer’s protocols. All measurements were performed based on the standard curves provided with the respective kits.

Hemolymph indices, such as TG (100020090), T-CHO (100020080), high-density lipoprotein cholesterol (HDL-C; 100020235), low-density lipoprotein cholesterol (LDL-C; 100020245) and albumin (ALB; 100020150), were analysed using kits (BioSino Bio-Technology & Science Inc., Beijing, China). In addition, the contents of alanine aminotransferase (ALT; C009-1-1), AKP, ACP, polyphenol oxidase (PPO; A136-1-1), T-AOC, glutathione (GSH; A006-2-1), glucose (GLU; A154-2-1), NOS, alanine aminotransferase (AST; C010-2-1) and MDA were determined using commercial kits (Nanjing Jiancheng Bioengineering Institute Co., Ltd., Nanjing, Jiangsu, China) and a microplate reader (SpectraMax M2) or an ultraviolet spectrophotometer (UV-1800). Quality control was ensured by including standard curves and control samples in each assay batch (Table S2).

2.5. Real-time quantitative PCR analysis

Total RNA (pooled samples from three shrimp/replicate) was extracted from the hepatopancreas using TRIzol reagent (RNAiso Plus 9108, Takara Biomedical Technology Co., Ltd., Dalian, Liaoning, China). The quality and integrity of the RNA samples were assessed using a spectrophotometer (NanoDrop 2000; Thermo Fisher Scientific Inc., Waltham, MA, USA) and by electrophoresis on a 1.2% agarose gel. Subsequently, RNA was reverse-transcribed into complementary DNA‌ (cDNA) using the HiScript II Q RT SuperMix kit (Vazyme Biotech Co., Ltd., Nanjing, Jiangsu, China). Transcript expression analysis was conducted via performing real-time quantitative PCR ChamQ SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd., Nanjing, Jiangsu, China) using a published amplification protocol method (Li et al., 2025). The PCR protocol comprised an initial denaturation (95 °C, 5 min), followed by 55 cycles of denaturation (95 °C, 10 s), annealing (58 °C, 10 s) and elongation (72 °C, 10 s). Subsequently, a melting curve analysis was performed with denaturation at 95 °C for 10 s, hybridisation at 65 °C for 10 s and final dissociation at 97 °C for 1 s, ending with a cooling step at 37 °C for 30 s. Gene-specific primers were designed using Primer Premier 3.0 based on the published L. vannamei sequences on National Center for Biotechnology Information (NCBI) and were validated by determining their amplification (Table S3). The transcript expression levels were calculated using the 2^–△△Ct method (Livak and Schmittgen, 2001). The β-actin gene was used for normalisation as it had been previously validated as a stable reference gene in the hepatopancreas of L. vannamei under various nutritional and physiological conditions (Chang et al., 2025; Mustafa et al., 2025; Shi et al., 2021; Sun et al., 2007).

2.6. Statistical analysis

The parameters related to growth performance and feed utilization were calculated as follows:

PWG (%) = (Average final body weight [FBW]– Average initial body weight [IBW])/Average IBW × 100;

SGR (%/d) = [ln (Average FBW) – ln (Average IBW)]/56 × 100;

HSI (%) = Hepatopancreas weight/FBW × 100;

CF (g/cm³) = Shrimp BW/Shrimp body length³ × 100;

FE = (Total FBW + Dead shrimp weight – Total IBW)/Dry feed intake;

FI (%/d) = Total feed intake/[(Total FBW + Dead shrimp weight – Total IBW)/2 × 56] × 100.

Data were expressed as mean and standard error of the mean (SEM). The assumptions of normality and homogeneity of variances were confirmed by performing the Shapiro-Wilk and Levene’s tests, respectively. After confirming that these assumptions were not violated, data were subjected to one-way analysis of variance (ANOVA) followed by Tukey’s test to identify significant differences (P < 0.05) among dietary treatments using IBM SPSS Statistic 26.0 (SPSS Inc., Chicago, IL, USA). Column charts were generated using Prism 10.0 (GraphPad software Inc., San Diego, CA, USA).

The mathematical model for ANOVA was as follows:

$$Y_{ij}=\mu +T_i+{\epsilon }_{ij}\text{,}$$

where Y_ij is the dependent variable; μ is the overall mean; T_i is the fixed effect of treatment with different dietary Mn sources (i = MnSO₄·H₂O, MnO₂, MnO₂NPs, Mn–Gly, and Mn–MHA); ε_ij is the random residual error.

3. Results

3.1. Growth performance and feed utilisation

As presented in Table 2, there was no significant effect on the growth performance (FBW, PWG, and SGR), feed efficiency, survival, or body indices (hepatosomatic index and condition factor) of L. vannamei among treatments after the 8-week feeding trial (P > 0.05).

Table 2.

Effects of different dietary Mn sources on growth performance and feed utilization of Litopenaeus vannamei.

Items	Dietary Mn sources1					SEM	P-value
	MnSO4·H2O	MnO2	MnO2NPs	Mn-Gly	Mn-MHA
IBW, g/shrimp	1.03	1.02	1.04	1.03	1.04	0.007	0.875
FBW, g/shrimp	16.26	16.73	16.05	16.56	16.77	0.116	0.220
PWG, %	1479.76	1544.07	1446.15	1500.59	1514.77	16.547	0.457
SGR, %/d	4.93	5.00	4.89	4.95	4.97	0.019	0.459
HSI, %	4.54	3.93	4.72	4.23	4.40	0.151	0.591
CF, g/cm3	0.98	0.90	0.95	0.96	0.94	0.012	0.384
FE	0.84	0.81	0.80	0.84	0.84	0.007	0.124
FI, %/d	3.75	3.84	3.90	3.70	3.73	0.028	0.067

IBW = initial body weight; FBW = final body weight; PWG = percent weight gain; SGR = specific growth rate; HSI = hepatosomatic index; CF = condition factor; FE = feed efficiency; FI = feed intake; SEM = standard error of the mean.

Within a row, means are not statistically significant difference at P > 0.05.

¹MnSO₄·H₂O, Mn sulfate; MnO₂, Mn dioxide; MnO₂NPs, Mn dioxide nanoparticles; Mn-Gly, Mn glycine chelate; and Mn-MHA, Mn chelate of hydroxy analogue of methionine.

3.2. Tissue Mn contents and transcript expression associated with Mn absorption and transport

As presented in Table 3, Mn deposition in the muscle and carapace of L. vannamei did not significantly differ among all dietary treatments (P > 0.05). However, the hepatopancreas Mn content was higher in the MnSO₄·H₂O, MnO₂NPs, Mn–Gly, and Mn–MHA groups than in the MnO₂ group (P = 0.002).

Table 3.

Effects of different dietary Mn sources on Mn deposition in Litopenaeus vannamei (mg/kg).

Items	Dietary Mn sources1					SEM	P-value
	MnSO4·H2O	MnO2	MnO2NPs	Mn-Gly	Mn-MHA
Muscle	3.52	3.55	3.62	3.75	3.81	0.047	0.204
Hepatopancreas	9.63a	8.37b	9.93a	10.09a	10.39a	0.240	0.002
Carapace	7.08	6.84	7.23	7.19	7.37	0.273	0.991

SEM = standard error of the mean.

Within a row, means without a common superscript letter differ at P < 0.05, n = 3.

¹MnSO₄·H₂O, Mn sulfate; MnO₂, Mn dioxide; MnO₂NPs, Mn dioxide nanoparticles; Mn-Gly, Mn glycine chelate; and Mn-MHA, Mn chelate of hydroxy analogue of methionine.

Owing to the notable differences in Mn deposition in the hepatopancreas, an analysis was conducted of the transcript expression associated with Mn transport and absorption. The results indicated that the mRNA levels of zip14, tmem165, and fpn1 were significantly higher in the shrimp fed with MnO₂NPs, Mn-Gly, and Mn-MHA diets than in those fed with MnO₂ diet (P < 0.05; Fig. 1). Moreover, the differences in the zip14, tmem165, and fpn1 mRNA expression between the MnSO₄·H₂O and MnO₂ groups were not statistically significant (P > 0.05).

Fig. 1.

Effects of different dietary Mn sources on the mRNA expression of absorption and transport in the hepatopancreas of Litopenaeus vannamei. MnSO₄·H₂O, Mn sulfate; MnO₂, Mn dioxide; MnO₂NPs, Mn dioxide nanoparticles; Mn-Gly, Mn glycine chelate; and Mn-MHA, Mn chelate of hydroxy analogue of methionine. Different lowercase letters above columns represent significant differences among treatments at P < 0.05 (n = 3).

3.3. Hemolymph biochemical parameters

As presented in Table 4, a consistent trend was observed for lipid metabolism parameters (TG, T-CHO, HDL-C, and LDL-C), with the most pronounced reduction occurring in the Mn-MHA group when compared with that in the other groups. The TG, T-CHO, and LDL-C levels were higher in the MnO₂-fed group than in the Mn-MHA-fed group (P < 0.05). In addition, the levels of HDL-C were lower in the Mn-MHA and MnO₂ groups than in the Mn-Gly group (P = 0.026). Moreover, GLU was significantly increased in the MnO₂ and Mn-Gly groups but was the lowest in the MnO₂NPs group (P = 0.005). Activity of ALT was significantly increased in the MnO₂ group than in the MnSO₄·H₂O and MnO₂NPs groups (P < 0.001). Activity of AST was higher in the MnO₂ group than in the Mn-MHA and MnSO₄·H₂O groups (P = 0.005). Activities of immune-related enzymes (AKP, ACP, and PPO) were significantly higher in the Mn-MHA than in the MnO₂ group (P < 0.05). Moreover, NOS activities were significantly higher in Mn-MHA than in the MnO₂NPs group (P = 0.009). In addition, ALB content was significantly higher in the MnO₂ than in the Mn-MHA group (P < 0.001). Regarding antioxidant status, shrimp fed with Mn-MHA demonstrated significantly higher activities of T-AOC and GSH-Px as well as GSH contents than those fed with MnO₂ (P < 0.05). Concurrently, the MDA levels were significantly lower in the Mn-MHA group than in the groups fed with inorganic Mn sources (P = 0.001).

Table 4.

Effects of different dietary Mn sources on serum biochemical parameters of Litopenaeus vannamei.

Items	Dietary Mn sources1					SEM	P-value
	MnSO4·H2O	MnO2	MnO2NPs	Mn-Gly	Mn-MHA
TG, mmol/L	1.10ab	1.16a	1.07ab	1.14a	0.86b	0.035	0.017
T-CHO, mmol/L	1.71ab	1.96a	1.50ab	1.61ab	1.38b	0.086	0.039
HDL-C, mmol/L	1.00ab	0.99b	1.01ab	1.09a	0.98b	0.013	0.026
LDL-C, mmol/L	1.64b	1.94a	1.54bc	1.44bc	1.34c	0.058	<0.001
GLU, mmol/L	5.84ab	6.19a	5.61b	6.07a	5.82ab	0.062	0.005
ALT, U/L	439.24c	551.01ab	456.86c	562.85a	490.24bc	14.226	<0.001
AST, U/L	371.06c	418.41a	401.80abc	403.25ab	386.91bc	4.975	0.005
ALB, g/L	10.44bc	11.47a	10.39bc	10.93ab	9.60c	0.180	<0.001
AKP, U/L	4.17a	3.09b	4.75a	4.57a	4.59a	0.187	0.004
ACP, U/L	6.40b	6.42b	8.18a	8.84a	8.67a	0.326	0.002
PPO, nmol/mL	51.41ab	44.84b	45.47b	45.08b	58.31a	1.768	0.029
T-AOC, U/mL	40.04ab	30.36b	38.98ab	38.84ab	47.47a	1.827	0.028
GSH-Px, U/mL	123.74b	124.51b	125.31b	128.29ab	136.95a	1.548	0.011
GSH, μmol/mL	42.99ab	34.10b	41.56ab	41.63ab	48.47a	1.590	0.043
NOS, U/mL	15.64ab	16.3ab	15.09b	16.56a	16.71a	0.192	0.009
MDA, nmol/mL	8.22ab	9.38a	8.20ab	7.08bc	6.26c	0.315	0.001

TG = triglyceride; T-CHO = total-cholesterol; HDL-C = high-density lipoprotein cholesterol; LDL-C = low-density lipoprotein cholesterol; GLU = glucose; ALT = aspartate transaminase; AST = alanine aminotransferase; ALB = albumin; AKP = alkaline phosphatase; ACP = acid phosphatase; PPO = polyphenol oxidase; T-AOC = total-antioxidant capacity; GSH-Px = glutathione peroxidase; GSH = glutathione; NOS = nitric oxide synthase; MDA = malondialdehyde; SEM = standard error of the mean.

Within a row, means without a common superscript letter differ at P < 0.05, n = 3.

¹MnSO₄·H₂O, Mn sulfate; MnO₂, Mn dioxide; MnO₂NPs, Mn dioxide nanoparticles; Mn-Gly, Mn glycine chelate; and Mn-MHA, Mn chelate of hydroxy analogue of methionine.

3.4. Hepatopancreas and intestinal biochemical parameters of L. vannamei

The hepatopancreas TG and T-CHO levels were significantly higher in shrimp fed with MnSO₄·H₂O diet than in those fed with MnO₂NPs and Mn-MHA diets (P < 0.05; Table 5). Conversely, the AKP, ACP, NOS, T-AOC, and GSH-Px activities were markedly enhanced in the Mn-MHA compared with the MnO₂ group (P < 0.05). Accordingly, the MDA content was significantly lower in the Mn-MHA group than in the groups fed with inorganic Mn sources (MnSO₄·H₂O, MnO₂, and MnO₂NPs) (P < 0.001).

Table 5.

Effects of different dietary Mn sources on biochemical parameters in the hepatopancreas of Litopenaeus vannamei.

Items	Dietary Mn sources1					SEM	P-value
	MnSO4·H2O	MnO2	MnO2NPs	Mn-Gly	Mn-MHA
TG, mmol/g prot	0.52a	0.40ab	0.32b	0.39b	0.36b	0.023	0.002
T-CHO, mmol/g prot	0.07a	0.05b	0.05b	0.05b	0.05b	0.002	0.015
AKP, U/g prot	29.68ab	23.58b	30.22ab	32.40a	31.63a	1.032	0.022
ACP, U/g prot	10.52bc	8.51c	13.04ab	12.60ab	15.14a	0.690	0.003
NOS, U/mg prot	0.78bc	0.61c	0.73bc	0.86b	1.19a	0.055	<0.001
T-AOC, mmol/g prot	1.58ab	1.38b	1.40b	1.77ab	2.13a	0.087	0.007
GSH-Px, U/mg prot	53.86ab	43.99b	51.45ab	62.15a	62.19a	2.042	0.001
MDA, nmol/mg prot	3.85ab	4.49a	3.83ab	2.85bc	2.35c	0.221	<0.001

TG = triglyceride; prot = protein; T-CHO = total-cholesterol; AKP = alkaline phosphatase; ACP = acid phosphatase; GSH-Px = glutathione peroxidase; MDA = malondialdehyde; SEM = standard error of the mean.

Within a row, means without a common superscript letter differ at P < 0.05, n = 3.

¹MnSO₄·H₂O, Mn sulfate; MnO₂, Mn dioxide; MnO₂NPs, Mn dioxide nanoparticles; Mn-Gly, Mn glycine chelate; and Mn-MHA, Mn chelate of hydroxy analogue of methionine.

The Mn–MHA group demonstrated significantly higher intestinal T-AOC activity than the other groups (P < 0.001; Table 6). And NOS activity was higher in the MnO₂NPs, Mn-Gly, and Mn-MHA groups than in the MnSO₄·H₂O and MnO₂ groups (P < 0.001).

Table 6.

Effects of different dietary Mn sources on T-AOC and NOS in the intestine of Litopenaeus vannamei.

Items	Dietary Mn sources1					SEM	P-value
	MnSO4·H2O	MnO2	MnO2NPs	Mn-Gly	Mn-MHA
T-AOC, mmol/g prot	0.88b	1.14b	0.79b	1.43b	2.36a	0.163	<0.001
NOS, U/mg prot	1.23b	1.05b	1.97a	2.18a	2.36a	0.151	<0.001

T-AOC = total-antioxidant capacity; prot = protein; NOS = nitric oxide synthase; SEM = standard error of the mean.

Within a row, means without a common superscript letter differ at P < 0.05, n = 3.

¹MnSO₄·H₂O, Mn sulfate; MnO₂, Mn dioxide; MnO₂NPs, Mn dioxide nanoparticles; Mn-Gly, Mn glycine chelate; and Mn-MHA, Mn chelate of hydroxy analogue of methionine.

3.5. Proximate composition analysis of whole shrimp and tissues

No significant differences were observed in the dry matter of the whole shrimp, and ash contents of hepatopancreas, moisture, and crude protein, and ash content of muscle (P > 0.05; Table 7). The crude lipid and protein contents of the whole shrimp were significantly lower in the Mn-MHA than in the MnSO₄·H₂O, MnO₂NPs, and Mn-Gly groups (P < 0.05). The ash content in the whole shrimp was significantly higher in the Mn-Gly than in the MnO₂ group (P = 0.002).

Table 7.

Effects of different dietary Mn sources on proximate composition in Litopenaeus vannamei (%, wet basis).

Items	Dietary Mn sources1					SEM	P-value
	MnSO4·H2O	MnO2	MnO2NPs	Mn-Gly	Mn-MHA
Whole shrimp
Dry matter	34.89	32.26	35.99	36.95	28.50	1.498	0.431
Crude protein	25.40a	20.06ab	24.54a	23.80a	19.08b	0.742	0.032
Crude lipid	6.87a	7.64a	7.11a	7.89a	5.21b	0.284	0.002
Ash	3.91ab	3.07c	3.47bc	4.46a	3.40bc	0.145	0.002
Hepatopancreas
Moisture	36.60a	35.91a	32.04b	33.31ab	33.07ab	0.561	0.011
Crude protein	17.61b	17.75b	19.25ab	19.97a	19.92a	0.314	0.003
Crude lipid	7.64a	7.14a	5.67ab	6.26ab	5.12b	0.277	0.002
Ash	1.00	1.01	1.02	1.04	1.00	0.007	0.643
Muscle
Moisture	26.35	26.37	25.23	25.38	24.41	0.354	0.394
Crude protein	21.33	21.35	21.10	21.32	21.30	0.254	0.999
Crude lipid	2.17a	2.23a	2.20a	1.84b	1.81b	0.052	<0.001
Ash	1.60	1.48	1.44	1.43	1.51	0.029	0.414

SEM = standard error of the mean.

Within a row, means without a common superscript letter differ at P < 0.05, n = 3.

¹MnSO₄·H₂O, Mn sulfate; MnO₂, Mn dioxide; MnO₂NPs, Mn dioxide nanoparticles; Mn-Gly, Mn glycine chelate; and Mn-MHA, Mn chelate of hydroxy analogue of methionine.

For the hepatopancreas, the crude protein content was significantly higher in the Mn-Gly and Mn-MHA groups than in the MnSO₄·H₂O and MnO₂ groups (P = 0.003). Contrarily, the crude lipid contents in the hepatopancreas were significantly higher in the MnSO₄·H₂O and MnO₂ groups than in the Mn-MHA group (P = 0.002). In addition, the moisture content was significantly higher in the MnSO₄·H₂O and MnO₂ groups than in the MnO₂NPs group (P = 0.011).

For the muscle, organic Mn supplementation (Mn-MHA and Mn-Gly) significantly reduced the crude lipid content compared with inorganic Mn sources (P < 0.001).

3.6. Transcript profiling of hepatic genes involved in antioxidant capacity and innate immune

The mRNA expression of gpx was significantly higher in the Mn-MHA group by 1.12-, 1.82-, 0.83-, and 0.86- fold than in the MnSO₄·H₂O, MnO₂, MnO₂NPs, and Mn-Gly groups, respectively (P < 0.001; Fig. 2A). Meanwhile, the mRNA expression of cat was highly upregulated in the MnSO₄·H₂O (0.42-fold), MnO₂NPs (1.14-fold), Mn-Gly (1.09-fold), and Mn-MHA (1.50-fold) groups compared with the MnO₂ group (P < 0.001). Furthermore, the organic Mn sources (Mn-Gly and Mn-MHA) exhibited higher sod mRNA expression than ones fed with the inorganic Mn sources (MnSO₄·H₂O, MnO₂, and MnO₂NPs) (P < 0.001).

Fig. 2.

Effects of different dietary Mn sources on antioxidant and non-specific immunity-related genes in the hepatopancreas of Litopenaeus vannamei. (A)The mRNA expression of antioxidant capacity. (B) The mRNA expression of non-specific immune. MnSO₄·H₂O, Mn sulfate; MnO₂, Mn dioxide; MnO₂NPs, Mn dioxide nanoparticles; Mn-Gly, Mn glycine chelate; and Mn-MHA, Mn chelate of hydroxy analogue of methionine. Different lowercase letters above columns represent significant differences among treatments at P < 0.05 (n = 3).

The lysozyme mRNA expression levels were most effectively upregulated by organic Mn sources, with Mn-MHA inducing the strongest response, followed by that of Mn-Gly (P = 0.002; Fig. 2B). The mRNA expression of nos increased by 0.55- to 2.16- fold in the Mn-Gly and Mn-MHA groups when compared with the MnSO₄·H₂O and MnO₂ groups (P = 0.028). The expression of acp increased by 0.65- to 0.88-fold in the Mn–MHA group when compared with the MnSO₄·H₂O, MnO₂, and MnO₂NPs groups (P = 0.005). The akp mRNA expression significantly increased by about 0.53- to 0.81-fold in the MnO₂NPs and Mn-Gly groups compared with the MnSO₄·H₂O and MnO₂ groups (P = 0.001). No significant differences in the po expression were observed among the groups (P = 0.219).

3.7. Transcript abundance of mRNA related to inflammation, apoptosis, and ERS

The transcript levels of pro-inflammatory genes, such as spz-5, rab6a, and il-16, was significantly suppressed in the Mn–MHA when compared with the inorganic Mn groups (MnSO₄·H₂O and MnO₂) (P < 0.05; Fig. 3A). In addition, the expression of tnf was significantly higher in the MnSO₄·H₂O and MnO₂ groups (by 1.03- and 1.23-fold, respectively) than in the Mn-MHA group (P < 0.001).

Fig. 3.

Effects of different dietary Mn sources on the inflammation- and apoptosis-related genes in the hepatopancreas of Litopenaeus vannamei. (A) The mRNA expression of inflammation. (B) The mRNA expression of apoptosis. MnSO₄·H₂O, Mn sulfate; MnO₂, Mn dioxide; MnO₂NPs, Mn dioxide nanoparticles; Mn-Gly, Mn glycine chelate; and Mn-MHA, Mn chelate of hydroxy analogue of methionine. Different lowercase letters above columns represent significant differences among treatments at P < 0.05 (n = 3).

The mRNA expression of p53 was significantly lower in the Mn-Gly and Mn-MHA groups than in the MnSO₄·H₂O, MnO₂, and MnO₂NPs groups (P = 0.001). In contrast, the mRNA expression of bl-1 exhibited an opposite trend to that of p53 (P = 0.002). Moreover, the transcript levels of caspase3 and caspase8 were significantly lower in the Mn-Gly and Mn-MHA groups than in the MnO₂ group (P < 0.05). Furthermore, the mRNA expression of sirt6 was significantly increased in the MnSO₄·H₂O and MnO₂ groups when compared with the organic Mn sources (Mn-Gly and Mn-MHA) and MnO₂NPs groups (P < 0.001).

Shrimp fed with Mn-MHA and Mn-Gly diets showed the lower expression levels of perk that activated the atf6 and eif2α when compared with those fed with MnO₂ diet (P < 0.05; Fig. 4). The atf4 mRNA expression level was significantly higher in the MnO₂ group (about 3.65-fold) than in the Mn-MHA group (P = 0.004).

Fig. 4.

Effects of different dietary Mn sources on mRNA expression of endoplasmic reticulum stress in the hepatopancreas of Litopenaeus vannamei. MnSO₄·H₂O, Mn sulfate; MnO₂, Mn dioxide; MnO₂NPs, Mn dioxide nanoparticles; Mn-Gly, Mn glycine chelate; and Mn-MHA, Mn chelate of hydroxy analogue of methionine. Different lowercase letters above columns represent significant differences among treatments at P < 0.05 (n = 3).

3.8. Transcript profiling of the hepatic genes involved in lipid metabolism

Similar to the lipid metabolism results from hepatopancreas and hemolymph biochemical data, the Mn–MHA group showed downregulated mRNA expression of fas, 6pgd, and acc 1, which were involved in lipid synthesis, and upregulated the expression of aco and cpt1, which were associated with lipolysis in the hepatopancreas, when compared with the MnO₂-fed group (P < 0.05; Fig. 5). In addition, the mRNA expression of fatp and fabp were significantly higher in the Mn–-MHA than in the MnO₂ group (P = 0.040), with no significant differences observed among the MnSO₄·H₂O, Mn-Gly, and MnO₂NPs groups (P > 0.05). The mRNA expression of srebp was higher in the MnO₂NPs than in the MnSO₄·H₂O group (P = 0.009). Moreover, no significant differences in srebp expression were observed among the MnO₂, Mn-Gly, and Mn-MHA groups (P > 0.05). Taken together, dietary Mn-MHA reduced lipid accumulation in the hepatopancreas by upregulating lipid lipolysis and transport.

Fig. 5.

Effects of different dietary Mn sources on mRNA expression of lipid metabolism in the hepatopancreas of Litopenaeus vannamei. MnSO₄·H₂O, Mn sulfate; MnO₂, Mn dioxide; MnO₂NPs, Mn dioxide nanoparticles; Mn-Gly, Mn glycine chelate; and Mn-MHA, Mn chelate of hydroxy analogue of methionine. Different lowercase letters above columns represent significant differences among treatments at P < 0.05 (n = 3).

4. Discussion

This study demonstrates that the different dietary Mn sources had no significant effect on the growth performance of juvenile L. vannamei. The optimal dietary Mn requirement for L. vannamei has been reported to be 30.69 mg/kg, when Mn-MHA is the source, and it should be within a range of 23.90 to 32.26 mg/kg (Cai et al., 2017; Li et al., 2025). In this study, the Mn levels in all five diets were sufficient to fulfill the optimal requirement of shrimp and thereby potentially mask the growth differences among treatments. The absence of growth differentials indicates that under Mn-sufficient conditions, growth performance is not a sensitive indicator of Mn source bioavailability. This finding contradicts with the finding of P. fulvidraco, in which organic Mn enhanced growth (Xu et al., 2023), but it remains consistent with the observations in Atlantic salmon (Salmo salar) (Prabhu et al., 2019). This disparity likely reflects species-specific metabolic responses and the saturation of growth requirements in L. vannamei, shifting the complex evaluation criteria to physiological biomarkers. The study demonstrates that the chemical form of Mn predominately influences L. vannamei at the physiological level and significantly modulates the antioxidant capacity, immune response, and metabolic homeostasis in the hepatopancreas.

Although growth remains unaffected, dietary organic Mn significantly increases Mn deposition in the hepatopancreas, with no statistical differences observed in the carapace and muscle. This tissue-specific deposition is closely associated with the tissue functional characteristics, Mn metabolic pathways, and chemical forms. It has been reported that the liver/hepatopancreas can regulate Mn homeostasis and that excessive Mn is preferentially deposited in the liver, with its net accumulation reflecting a balance between uptake and efflux of Mn (Liu et al., 2021). In addition, the mRNA expression of Mn transporter (zip14, tmem165, and fpn1) is significantly upregulated in shrimp fed with organic Mn and MnO₂NPs diets. Zip14 is a Mn²⁺ bicarbonate symporter and transporter (Pinilla et al., 2011). It is most abundantly expressed in the liver, and zip14 is located at the basolateral membrane of hepatocytes (Nam et al., 2013). The FPN1 mediates Mn transmembrane transport through its metal efflux capacity (Madejczyk and Ballatori, 2012), while TMEM165 highly affects the systemic Mn homeostasis by modulating intracellular Mn balance (Liu et al., 2021). Therefore, the results indicate that organic Mn exhibits higher bioavailability than inorganic Mn by primarily accumulating in the hepatopancreas to regulate Mn homeostasis based on Mn deposition in tissues and the expression of genes related to Mn absorption and transport. Notably, this increased Mn accumulation up to a safe and beneficial range, as evidenced by the concomitant physiological improvement observed in the same organ as well as significantly physiological improvements observed in the hepatopancreas, which includes the enhancement of antioxidant enzyme activities (e.g. T-AOC and GSH-Px) and reduced lipid peroxidation (MDA), all of which are incompatible with a state of Mn overload. The comparable effects of MnO₂NPs can be attributed to the high specific surface area of the nanomaterials, which further enhances the solubility and bioavailability of Mn and thereby activates the metabolic efficacy of the transport system (Noventa et al., 2018). These results indicate that different Mn forms establish distinct bioavailability pathways by specifically modulating metal transporter proteins.

Mn, being an important antioxidant trace element, has involvement in many biological functions (Aguirre and Culotta, 2012). Previous studies have shown that dietary Mn levels influence the activities of ACP, AKP, NOS, and GSH-PX as well as levels of GSH, MDA, and T-AOC (Cao et al., 2022; Jia et al., 2020; Liu et al., 2023; Xu et al., 2023; Zhang et al., 2024). Herein, organic Mn sources significantly improved antioxidant capacity and immune response compared with inorganic Mn sources. A previous study on beluga sturgeon (Huso huso) reported higher bioavailability and stronger antioxidant efficacy for organic trace element (Mohseni et al., 2021). In P. fulvidraco, dietary Mn-Gly and Mn-MHA can increase antioxidant capacity and reduce oxidative stress in the intestine (Xu et al., 2023). Moreover, organic Mn sources significantly increase the expression levels of antioxidant-related genes (gpx, cat, and sod) as well as innate immunity-related genes (nos, acp, and akp) in the hepatopancreas, with the most pronounced effects relative to the MnO₂ group. Song et al. (2025) suggested that Mn-MHA supplementation can effectively alleviate the immune response of L. vannamei induced by the replacement of fish meal with high Clostridium autoethanogenum protein. In summary, relative to the inorganic Mn sources, organic Mn sources exert a more pronounced effect on the improvement of the antioxidant and innate immunological capabilities of L. vannamei.

Endoplasmic reticulum stress is a pivotal mechanism for maintaining cellular homeostasis, mainly through the PERK-eIF2α, ATF6, and IRE1-XBP1 pathways of the unfolded protein response (Cao and Kaufman, 2014). Perk suppresses global protein synthesis by phosphorylating eIF2α and selectively upregulates the expression of stress-response genes controlled by ATF4 by forming a central regulatory network that allows cells to counteract protein homeostasis disruptions (Walter and Ron, 2011; Zhao et al., 2022). In the study, the expression of ERS marker genes (eif2α, perk, atf6, and atf4) are markedly lower in the organic Mn source groups (Mn-MHA and Mn-Gly) than in the inorganic Mn source groups, suggesting that the former significantly enhance cellular antioxidant defence capabilities (Xu et al., 2023). A closer examination of the underlying mechanisms reveals a significant interplay between ERS and oxidative stress within the integrated stress response. Oxidative stress inhibition can effectively mitigate ERS (Taniuchi et al., 2016; van’t Wout et al., 2014). The superior antioxidant effects of organic Mn are likely attributable to its high bioavailability. Its chelated structure allows for bonded mineral to be carried into the mucosal cell and absorbed as an intact molecule and thereby minimises antagonisms and ensures a highly efficient delivery to tissues (Ashmead, 1993). The increased bioavailability of organic Mn sources, a well-established phenomenon (Nie et al., 2016), is supported in the study by the higher Mn accumulation observed in the hepatopancreas of the shrimp fed with organic Mn diets. These findings suggested that this enhanced tissue Mn delivery ensures a more sufficient supply for the biosynthesis and activity of key antioxidant enzymes, such as Mn-superoxide dismutase (Grujicic and Allen, 2025; Nie et al., 2016). This mechanism provides a convincing explanation for the central finding: despite equivalent growth performance, organic Mn sources confer substantial advantages by enhancing antioxidant capacity and attenuating cellular oxidative damage and ERS.

Apoptosis is a programmed cell death process regulated by complex molecular signals (Obeng, 2021). In the study, different dietary Mn sources significantly regulated the expression of apoptotic genes. The Sirt6 can promote apoptosis by decreasing Bcl-2 expression and increasing Bax expression. Cytochrome c forms an apoptosome with apoptotic protease-activating factor 1, which is followed by the activation of Caspase3 to execute apoptosis (Beroske et al., 2021; Chen et al., 2018; Zhra et al., 2024). In addition, Fas ligand binds to its receptor on the surface of target cells and induces the apoptosis signalling pathway, which results in the activation of pro-Caspase8, which in turn initiates the cascade activation of Caspase3 and ultimately induces apoptosis (Abou-Ghali and Stiban, 2015). Furthermore, p53, as a classic tumour-suppressor, can directly induce apoptosis via transcriptional activation of pro-apoptotic genes or modulation of mitochondrial membrane permeability (Basu and Haldar, 1998; Ma and Yang, 2016). The ERS-related protein Bl-1 exerts an anti-apoptotic effect by inhibiting Bax translocation (Henke et al., 2011). Compared with inorganic Mn diets, organic Mn diets (Mn-Gly and Mn-MHA) significantly upregulate the expression of the anti-apoptotic bl-1 and downregulate the pro-apoptotic genes (sirt6, p53, caspase3, and caspase8) when compared with inorganic Mn diets. This suggests that organic Mn sources can effectively inhibit apoptotic process, which is consistent with the finding of Liu et al. (2020), who reported that dietary organic Mn reduces ERS-triggered apoptosis by activating autophagy. This suggests the need to precisely regulate the supplemental dose of organic Mn sources to balance its dual effects of promoting and inhibiting apoptosis in practical applications. In addition, some studies report that high Mn concentration can induce oxidative stress, elevate ROS, activate the PERK/MAPK signalling pathway, and exacerbate apoptosis (Ding et al., 2020).

Inflammation is a crucial immune defence mechanism. Pro-inflammatory genes, such as il-16, tnf, spz, and rab6a, regulate inflammatory mediator synthesis, immune cell recruitment, and signalling pathway activation (Micaroni et al., 2013; Newton and Dixit, 2012; Slaats et al., 2016). Manganese can effectively alleviate oxidative stress–mediated inflammatory responses by enhancing SOD activity and reducing ROS (Mathieu et al., 2017; Zhang et al., 2020). In the study, the shrimp fed with organic Mn sources (Mn-MHA and Mn-Gly) demonstrate considerably lower expression of pro-inflammatory factors (il-16, tnf, spz, and rab6a) than those fed with inorganic Mn source. Xu et al. (2023), highlighting the importance of optimising Mn source in preventing inflammatory diseases in P. fulvidraco. Therefore, organic Mn sources exhibit high regulatory efficiency in suppressing inflammatory responses, which potentially provide a new nutritional strategy for managing inflammation-related disorders in aquatic species.

Manganese influences lipid metabolism mainly through its role as a cofactor for enzymes central to this process (Xu et al., 2023; Zhao et al., 2024). The study demonstrates a striking contrast between Mn sources: Mn-MHA supplementation significantly reduces crude lipid content in whole shrimp, hepatopancreas, and muscle, as evidenced by real time quantitative PCR results related to the downregulation of lipid synthesis and upregulation of lipolysis gene. The superior efficacy of organic Mn-MHA in modulating lipid metabolism, compared with inorganic sources, can be attributed to its higher bioavailability, as evidenced by higher hepatopancreatic Mn deposition. These finding suggest that the enhanced Mn delivery to the hepatopancreas, the central metabolic organ, more effectively activates or supports Mn-dependent metabolic pathways. One plausible mechanism is that the enhanced energy production from increased mitochondrial β-oxidation (as evidenced by upregulated aco and cpt1) alters the cellular energy status, which can activate energy-sensing pathway via inhibiting acetyl-CoA carboxylase and promoting fatty acid oxidation. This contrasts with the findings of Xu et al. (2023), who reported increased lipid deposition in P. fulvidraco with organic Mn-based diet. This discrepancy highlights a fundamental species-specific difference in the way crustaceans and teleost fish regulate energy metabolism in response to trace minerals with high-bioavailability.

The findings of this study have direct implications for sustainable shrimp feed formulation. The superior bioavailability and functional benefits of organic Mn sources (Mn-Gly and Mn-MHA) make them ideal candidates for premium aquafeeds aimed at enhancing stock resilience. By improving antioxidant capacity, immune response, and hepatopancreatic health, the inclusion of these organic Mn sources in diets can reduce disease outbreaks and the reliance on antibiotics in commercial farming. Moreover, the ability of Mn-MHA to modulate lipid metabolism and reduce hepatopancreas lipid deposition is a strategy for improving metabolic health and feed efficiency. Therefore, the adoption of these functional Mn sources is consistent with the goal of improving shrimp health and productivity while promoting a comparatively sustainable and environmentally responsible aquaculture.

5. Conclusion

In conclusion, feeding L. vannamei with organic Mn sources elicits more pronounced effects at physiological and metabolic levels than growth, relative to when fed with inorganic Mn sources. In the study, organic Mn supplementation increased Mn accumulation as well as enhanced Mn absorption and transport in the hepatopancreas. Furthermore, Mn-MHA and Mn-Gly enhanced antioxidant and immune capacity as well as downregulated genes related to inflammation, apoptosis, and ERS. In addition, Mn-MHA regulated lipid metabolism by reducing lipid synthesis and promoting lipolysis. Therefore, organic Mn sources (Mn-Gly and Mn-MHA) were recommended as optimal Mn supplements for L. vannamei.

Credit Author Statement

Hongyu Peng: Writing – original draft, Visualization, Software, Methodology, Conceptualization. Shuqin Li: Writing – original draft, Conceptualization. Min Jin: Supervision, Resources, Methodology. Lu Zhang: Supervision, Software, Resources. Jinlin Wang: Software, Resources. Yu He: Software, Resources. Xiaoru Chen: Supervision, Software, Resources. Yinzhao Zhang: Supervision, Software, Resources. Feng Tang: Supervision, Software, Resources. Peng Sun: Writing – review & editing, Supervision, Resources, Methodology, Funding acquisition. Qicun Zhou: Writing – review & editing, Supervision, Resources, Methodology, Funding acquisition.

Declaration of competing interest

We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, and there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the content of this paper. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Lu Zhang and Xiaoru Chen are currently employed by Tongwei Agricultural Development Co., Ltd. (Chengdu, Sichuan, China); Yinzhao Zhang and Feng Tang are currently employed by Zhejiang Fengyu Marine Organism Products Co., Ltd. (Zhoushan, Zhejiang, China).

Appendix A. Supplementary data

The following is the Supplementary data to this article: