Leonardite humic acid enhances growth, lipid utilization and antioxidant status in juvenile Penaeus vannamei

1 Introduction

Aquaculture is a vital source of sustainable, nutritious, and affordable animal protein (Satheesh et al., 2023). The vertical and horizontal expansion of aquaculture can substantially increase production. In this context, cultivating commercially important euryhaline fish and shrimp species in under-utilized inland saline water (ISW) can play a pivotal role (Talukdar et al., 2021; Jana et al., 2021a; Paul et al., 2022a, b; Debroy et al., 2022; Jana et al., 2022; Paul et al., 2023; Bhusare et al., 2023; Pandey et al., 2024; Paul et al., 2024). Accordingly, white-leg shrimp, Penaeus vannamei has emerged as a preferable species for inland saline aquaculture because of its high growth rate, short production cycle (4 months), good market demand and value, less susceptibility to diseases and wide range of salinity tolerance (0.5-51 ppt) (Jana et al., 2021a; Chuphal et al., 2021; Panmei et al., 2023; Raghuvaran et al., 2023). However, feeding of high dietary protein will less availability of non-protein energy sources (lipid and carbohydrate) not only enhances the cost of feed, but also pollute the cultured water in terms of nitrogenous waste (Jana et al., 2021b), because high amount of dietary protein derived amino acids undergo energy production to satisfy energy demand at the compromising of growth and its subsequent deterioration due to ammonia stress (Davis et al., 1993; Jana et al., 2021a; Das et al., 2023). To overcome this situation, the concept of dietary protein sparing effect of lipid and carbohydrate needs to be addressed to produce cost-effective and eco-friendly feed with less carbon footprint in aquaculture (FAO, 2022). From this perspective, the use of low protein and high energy/lipid (LPHE/LPHL) diet can be the crucial to sustain different aquaculture production systems (Das et al., 2023). However, shrimps can utilize dietary lipid less efficiently due to poor secretion of bile acid and thus higher dietary lipid can cause metabolic stress concomitant with growth retardation and excess fat deposition in body of shrimp (Glencross, 2009; Jannathulla et al., 2019; Chuphal et al., 2021; Jaffer et al., 2024). Supplementation of LPHE/LPHL diets with nutraceuticals that enhance lipid utilization and alleviate lipid-induced stress may improve growth performance, health status, and sustainable shrimp production. In this context, use of humates in fish feed are gaining momentum for growth enhancement of fish (Zhang, 2018; Jusadi et al., 2020).

Recent studies highlight leonardite-derived humic acid (LHA) as a promising functional feed additive that enhances growth, nutrient utilization, antioxidant capacity, immune response, and intestinal health in shrimp aquaculture (Raghuvaran et al., 2023; Bhusare et al., 2023; Panmei et al., 2023). While the benefits of humic substances, such as sodium humate, fulvic acid, and humic acid have been well-documented in finfish and crustaceans (Liao et al., 2022; Rasidi et al., 2021). Humic substances (HSs) are complex compounds and their dietary inclusion protects intestine mucosa (Aksu and Bozkurt, 2009) with improving nutrient digestibility and retention (Taklimi et al., 2012). Dietary microbial humic acid (HA) enhances gastrointestinal health and nutrient utilization through multiple mechanisms. Owing to its colloidal structure, HA can adsorb toxins and harmful metabolites while forming a protective layer over the intestinal mucosa, thereby reducing epithelial exposure to pathogens. Its antioxidant, anti-inflammatory, and metal-chelating properties help maintain intestinal barrier integrity and reduce oxidative stress. Furthermore, HA has been reported to improve intestinal morphology by increasing villus height and preserving mucosal integrity, thereby enhancing nutrient digestion and absorption and thus feed utilization (Ji et al., 2006; Taklimi et al., 2012). HA’s phenolic and carboxylic groups provide strong antioxidant effects by scavenging free radicals and reducing oxidative stress. It also binds to toxins and pathogens, preventing their absorption, and disrupts microbial cell membranes that support overall animal health (Gomez de Melo et al., 2016; Mao, 2019). Leonardite humic acid (LHA) (Taklimi et al., 2012). Leonardite humic acid (LHA) has been reported to influence intermediary metabolism by modulating the activity of key enzymes involved in lipid and carbohydrate metabolism viz., carnitine palmitoyltransferase-1 (CPT-1), the rate-limiting enzyme responsible for transport of long-chain fatty acids into mitochondria for β-oxidation, thereby promoting lipid catabolism and energy production. LHA has also been associated with increased activity of glucose-6-phosphate dehydrogenase (G6PDH), the first and rate-limiting enzyme of the pentose phosphate pathway, which generates NADPH required for reductive biosynthesis and maintenance of cellular antioxidant defenses. The coordinated regulation of CPT-1 and G6PDH suggests that LHA improves metabolic flexibility by enhancing lipid utilization while maintaining cellular redox homeostasis, thereby supporting improved growth performance, feed efficiency, and stress resilience in aquatic animals (Taklimi et al., 2012; Yilmaz et al., 2018; Lieke et al., 2021). The combined antioxidant, protective, and metabolic effects of HA lead to improved feed conversion ratios (FCR) and overall growth when administered through feed or water (Gomez de Melo et al., 2016). Dietary humic acid can improve the growth and non-specific immunity of Nile tilapia (Jusadi et al., 2020), red swamp crayfish, Procambarus clarkia (Zhang, 2018), white shrimp, Litopenaeus vannamei (Fierro-Coronado et al., 2018) and loach (Gao et al., 2018). Moreover, dietary humic acid modulates the animal’s metabolism with better growth performance (Wang et al., 2008; Aeschbacher et al., 2012). Gao et al. (2018) demonstrated that dietary fulvic acid at 0.9-1.2% enhances growth and feed efficiency, with 1.2% producing the strongest antioxidant response in juvenile P. vannamei.

HSs, including HA, have shown considerable potential as functional feed additives by improving growth, feed utilization, intestinal health, antioxidant capacity, and immune responses in fish. However, information on their dietary inclusion in shrimp remains limited. Although studies in P. vannamei have demonstrated beneficial effects of humic substances on growth, nutrient utilization, and health, the underlying physiological mechanisms remain incompletely understood. Therefore, the present study investigated the effects of leonardite humic acid supplementation on growth, nutrient utilization, digestive and metabolic enzyme activities, and antioxidant responses in juvenile P. vannamei fed diets with reduced protein and increased lipid levels under inland saline water conditions.

2 Materials and methods

2.1 Ethical declarations

After approval from the concerned committee of ICAR-Central Institute of Fisheries Education (ICAR-CIFE), Mumbai, India, the present study was conducted with recommended guidelines for animal use.

2.2 Experimental animals

The present study was conducted at wet laboratory of ICAR-CIFE, Rohtak Centre, Haryana, India. The juvenile white-leg shrimp, P. vannamei (Boone 1931) obtained from the Baniyani Farm of Rohtak Centre were transported to the wet lab in polythene bags with oxygenated ISW (10 ppt salinity) and then transferred to two circular tanks (1.14 m dia,1.00 m depth,1000 L capacity, 770 L water volume). Afterward, the stock was acclimated for 15 days with K⁺ fortified ISW of 10 ppt salinity. The required concentration of water K⁺ was calculated according to Davis et al. (2005) using the formula of {(10.7 × desired salinity) – available K⁺ in ISW} and fulfilled through applying KCl. Round the clock aeration was provided to the tanks and the shrimps were fed with commercial feed (360 g/kg crude protein, CP and 50 g/kg lipid) four times daily at satiation level during acclimation period. At the same time, K⁺ fortified ISW was stored in few other cemented storage tanks and used for filling the experimental tanks as and when necessary, during the entire experimental period.

2.3 Formulation and preparation of the experimental diets

Following the 3×3 factorial design (three levels of protein-lipid, 3 levels of LHA), each of three formulated hetero-nitrogenous (around 300-360 g/kg CP), hetero-lipidic (50-90 g/kg) and hetero-caloric (374-398 Kcal digestible energy/100g) semi-purified diets was supplemented with 0, 9 and 12 g/kg leonardite humic acid, LHA at the cost of cellulose. Accordingly, nine experimental diets such as P₃₆L₅LHA₀ (360 g/kg CP, 50 g/kg lipid and 0 g/kg LHA), P₃₆L₅LHA_0.9 (P₃₆L₅LHA₀ diet supplemented with 9 g/kg LHA), P₃₆L₅LHA_1.2 (P₃₆L₅LHA₀ diet supplemented with 12 g/kg LHA), P₃₃L₇LHA₀ (330 g/kg CP, 70 g/kg lipid and 0 g/kg LHA), P₃₃L₇LHA_0.9 (P₃₃L₇LHA₀ diet supplemented with 9 g/kg LHA), P₃₃L₇LHA_1.2 (P₃₃L₇LHA₀ diet supplemented with 12 g/kg LHA), P₃₀L₉LHA₀ (300 g/kg CP, 90 g/kg lipid and 0 g/kg LHA), P₃₀L₉LHA_0.9 (P₃₀L₉LHA₀ diet supplemented with 9 g/kg LHA), P₃₀L₉LHA_1.2 (P₃₀L₉LHA₀ diet supplemented with 12 g/kg LHA) were formulated (Table 1) and prepared. According to the formula, all the finely mashed ingredients were weighed and then except oils and additives, other weighed ingredients were mixed uniformly in clear plastic trays and hydrated with the required amount of luke warm water and mixed to form a dough. Then, the dough was put in a heat-resistant plastic bag and steam cooked in a pressure cooker for 20 mins. After cooling, the cooked dough was dispersed in a tray and the oils and additives (LHA, Humate India Private Limited, CAS No. 1415-93-6, lecithin, cholesterol, butylated hydroxyl toluene, vitamin-mineral mixture, betaine, vitamin E and stay C) were added to it, mixed uniformly and again a new dough was made. The dough was then pressed through the mechanical pelletizer (S.B. Panchal & Co. Ltd, India) fitted with a 1 mm diameter perforated die to make the pellets. The pellets were then dried at room temperature for one day followed by oven drying at 42 °C until achieving a moisture level just below 10%. Dried pellets were then broken into small pieces approximately 4-6 mm in length, packed in airtight zipper bags, and stored at 4 °C until used for feeding (Jana et al., 2022).

Table 1. Formulation and proximate composition of experimental diets.

Proximate composition is expressed as means of triplicate (n=3); Fish meal (652.30 g/kg crude protein, CP, 92.21 g/kg Ether extract, EE); Shrimp meal (328.20 g/kg CP, 46.20 g/kg EE); Soybean meal (532.90 g/kg CP, 15.20 g/kg EE); Groundnut oil cake (462.40 g/kg CP, 94.80 g/kg EE); Wheat flour (91.70 g/kg CP, 11.80 g/kg EE); Maize flour (83.90 g/kg CP, 40.30 g/kg EE); Broken rice (10.51 g/kg CP, 12.80 g/kg EE).

P₃₆L₅LHA₀, 360 g/kg crude protein, 50 g/kg lipid and 0 g/kg leonardite humic acid (LHA); P₃₆L₅LHA_0.9, P₃₆L₅LHA₀ diet supplemented with 9 g/kg LHA; P₃₆L₅LHA_1.2, P₃₆L₅LHA₀ diet supplemented with 12 g/kg LHA; P₃₃L₇LHA₀, 330 g/kg crude protein, 70 g/kg lipid and 0 g/kg LHA; P₃₃L₇LHA_0.9, P₃₃L₇LHA₀ diet supplemented with 9 g/kg LHA; P₃₃L₇LHA_1.2, P₃₃L₇LHA₀ diet supplemented with 12 g/kg LHA; P₃₀L₉LHA_0, 300 g/kg crude protein, 90 g/kg lipid and 0 g/kg LHA; P₃₀L₉LHA_0.9, P₃₀L₉LHA₀ diet supplemented with 9 g/kg LHA; P₃₀L₉LHA_1.2, P₃₀L₉LHA₀ diet supplemented with 12 g/kg LHA.

BHT, butylated hydroxytoluene;

Composition of vitamin-mineral mixture (PRE-MIX PLUS) (quantity/kg): Vitamin A, 5,500,000 IU, Vitamin D3, 1,100,000 IU, Vitamin B2, 2,000 mg, Vitamin E, 750 mg, Vitamin K, 1,000 mg, Vitamin B6, 1,000 mg, Vitamin B12, 6 mg, Calcium Pantothenate, 2,500 mg, Nicotinamide, 10 g, Choline Chloride, 150 g, Mn, 27,000 mg, I, 1,000 mg Fe, 7,500 mg, Zn, 5,000 mg, Cu, 2,000 mg, Co, 450 mg, L-lysine, 10 g, DL-Methionine, 10 g, Selenium, 125 mg, Vitamin C, 2,500 mg.

CMC, carboxymethyl cellulose;

Stay C, protected vitamin C;

Vit. E, vitamin E;

LHA, leonardite humic acid (Humate India Private Limited, CAS No (1415-93-6); ⁸GE, gross energy;

DE, digestible energy;

P/E, protein to energy ratio;

CP, crude protein.

2.4 Experimental design, setup and feeding trial

Following a 3×3 factorial design, 27 circular fiber-reinforced plastic tanks (1 m depth, 0.8 m diameter, 500 L capacity, 400 L water volume) were used for feeding trials. The tanks were initially acid washed, filled with potassium permanganate solution (4 mg/L), left overnight, flushed out next morning, subsequently washed thoroughly with clean water and dried well under bright sunlight for the whole day. Then the tanks were filled with K⁺ fortified ISW of 10 ppt salinity from storage tanks and provided with round-the-clock aeration. Six hundred seventy five (675) acclimated P. vannamei juveniles (avg. body wt. 5.02 ± 0.01 g) were randomly distributed into nine experimental groups (P₃₆L₅LHA₀, P₃₆L₅LHA_0.9, P₃₆L₅LHA_1.2, P₃₃L₇LHA₀, P₃₃L₇LHA_0.9, P₃₃L₇LHA_1.2, P₃₀L₉LHA₀, P₃₀L₉LHA_0.9 and P₃₀L₉LHA_1.2) in triplicate with a stocking density of 25 shrimps per tank (62.5 nos./m^3,following 60 nos./m³, as recommended by Coastal Aquaculture Authority of India). The water salinity was maintained at 10 ppt throughout the experimental period. Shrimps were offered experimental feed at 4% of tank biomass per day, divided into four equal meals (7:30, 13:30, 18:30, and 23:30 h) daily throughout the experimental period of 60 days. The feces were siphoned out every morning before starting the next day’s feeding, and an equal volume of siphoned water was replenished with ISW of same salinity. The weight of shrimps from each tank were measured fortnightly to adjust the ration size. Throughout the experimental period, 30% of water from each experimental tank was exchanged with an equal volume of ISW of same salinity at the interval of every three days. Total amount of experimental feed provided was recorded to calculate dry matter intake and subsequently feed utilization metrics. Daily shrimp mortality of each tank was monitored throughout the experimental period to calculate the survival rate.

2.5 Physico-chemical parameters of water

The water temperature, pH, dissolved oxygen (DO) and salinity of all the experimental tanks were monitored daily by using a water thermometer (Aquaworld, India), a pH meter (Aquaworld, India), DO meter (Aquaworld, India) and refractometer (Essico, India), respectively. Following titrimetric method (APHA, 2005), total hardness, total alkalinity, free carbon dioxide and ionic concentrations of calcium (Ca²⁺) and magnesium (Mg²⁺) were determined at 3 days’ interval. The potassium ion (K⁺) concentration was estimated by using a flame photometer (Electrostatic, India). The ammonia-N, nitrite-N and nitrate-N concentration was determined at three days’ interval using ammonia-nitrite test kit in spectrophotometer (Spectro quant NOVA-MERCK, Germany) and a vapor pressure osmometer (VAPRO^®, Germany) was used to estimate the water osmolality of each experimental tank.

2.6 Sampling

The initial and final body weight of shrimp were measured using an electronic weighing balance (accuracy ±0.01 g). At the end of trial, feed was restricted for overnight and followed by five shrimps were randomly collected from each tank for the whole-body proximate composition analysis. Another four shrimps were collected from each tank, anesthetized in ice-chilled water (Jana et al., 2021a) and subjected to the collection of haemolymphs in 1.5 ml eppendorf tubes, which were then refrigerated (4 °C) for 15 min. Then the clot was macerated by a rod and centrifuged in a cooling centrifuge machine (Thermo Scientific) at 3000 g for 15 min. Finally, the supernatant (serum) was collected in another eppendorf tubes and stored at -40 °C until used for analysis of serum parameters. For hemocyanin estimation, another four shrimps from each tank were subjected to haemolymph collection in cold shrimp anticoagulant (SIC-EDTA, NaCl 450 mM, KCl 10 mM, Hepes 10 mM, EDTA 10 mM, pH 7.3) containing eppendorf tubes. The same shrimps were then dissected to obtain the gill and hepatopancreas. The weight of the respective hepatopancreas and shrimp was measured to calculate the hepatopancreas-somatic index (HPSI). Immediately after collection, the gill and hepatopancreas tissues were homogenized.

2.7 Growth, feed and nutrient utilization, body indices and survival

The calculation was made using the following formulae.

WG(g)=Final wet weight (g)−Initial wet weight

SGR (%/day)=lnof final wet weight−lnof initial wet weightExperimental period (days)×100

FCR=Feed intake (dry weight in g)body weight gain (wet weight in g)

PER=Wet weight gain (g)Protein intake (dry weight in g)

LER=Wet weight gain (g)Lipid intake (dry weight in g)

HPSI (%)=Wet weight of Hepatopancreas (g)Wet weight of shrimp (g)×100

Survival (%)=Total no. of shrimp harvestedTotal no. of shrimp stocked×100

2.8 Proximate analysis of diets and whole body of shrimp

The proximate composition of experimental diets and the whole body of shrimps were determined using standard methods (AOAC, 2005). A hot air oven was used to estimate moisture content. The rest of proximate components were analyzed on dry matter basis. The crude protein (CP) content was estimated by micro-kjeldahl assembly (Kelplus kjeldahl system, PELICAN, India) and the ether extract (EE) or lipid content was determined by a Soxhlet apparatus (PELICAN, India). The fat-free samples were subjected to acid and alkali digestion in fibroton (Tulin Equipments, India) followed by ignition of sample in a muffle furnace at 550 °C for 6 h to estimate crude fiber (CF). The total ash (TA) content was estimated by incinerating the sample in a muffle furnace at 550 °C for 6 h. Nitrogen-free extract (NFE) of diets and total carbohydrate (TC) of shrimp samples were calculated by the subtraction method using the following formulae.

NFE (%)={100−(CP%+EE%+CF%+TA%)}

TC (%)={100−(CP%+Lipid%+TA%)}

Finally, the proximate components except water of shrimp samples were expressed on wet weight basis.

A bomb calorimeter (Changshu Instruments, China) was used to estimate gross energy (GE) of diets. The digestible energy (DE) content (Halver, 1976) and protein-to-energy ratio (P: E) of diets were calculated by the following formulae.

DE (Kcal/100 g)={(CP%×4)+(EE%×9)+(NFE%×4)}

P: E={(CP%×1000)/DE(Kcal/100 g)}

2.9 Enzyme assays

2.9.1 Tissue homogenate preparation

Teflon-coated mechanical homogenizer (REMI Equipment, India) was used to prepare 5% tissue homogenates of gill and hepatopancreas in ice-cold phosphate buffer (0.025 M KH₂PO₄, 0.025 M Na₂PO₄.12H₂O, pH 7.5) in glass tubes. The cold chain was maintained throughout the whole procedure to preserve enzymatic activity. Then a refrigerated centrifuge machine (Heraeus Megafuge, Thermo Fisher Scientific, Germany) to centrifuge the tissue (gill and hepatopancreas) homogenates at 10,000 g for 10 min and the supernatants were collected in 2 ml eppendorf tubes and stored at −40 ⁰C until used for enzyme assays.

2.9.2 Tissue protein estimation

The total protein of different tissue homogenates was determined by the Bradford method (Bradford, 1976), and the resultant tissue protein value was used to calculate enzyme activity.

2.9.3 Activities of digestive enzymes

DNS (3, 5-di-nitro salicylic acid) method (Rick and Stegbauer, 1974) was employed to estimate the amylase activity and expressed as a micromole of maltose released/min/mg protein. The casein digestion method (Drapeau, 1976) quantified the protease activity and expressed as micromole of tyrosine released/min/mg protein. Lipase activity was assayed using the titrimetric method of Cherry and Crandall (1932) and expressed as units/h/mg protein.

2.9.4 Activities of lipid metabolic enzymes

According to Zheng et al. (2013) and DeMoss and Gibbs (1955), the carnitine palmitoyltransferase 1 (CPT-1) and glucose-6-phosphate dehydrogenase (G₆PDH) activities of hepatopancreas were analyzed and expressed as mM DTNB converted/min/mg protein and mM phosphorus released/min/mg protein, respectively.

2.9.5 Activities of oxidative stress enzymes

Superoxide dismutase (SOD) activity was assayed according to the method described by Misra and Fridovich (1972), whereas Catalase (CAT) activity was determined by the method described by Takahara et al. (1960). The activities of SOD and CAT were expressed as 50% inhibition of epinephrine autooxidation/mg protein/min and nanomoles H₂O₂ decomposed/mg protein/min, respectively.

2.10 Hepatopancreatic malondialdehyde

Hepatopancreatic MDA value was estimated according to Sharma and Murti (1968) and calculated as follows:

MDA (Nanomole/mg protein) = (O.D./192.305 M)/cm.

2.11 Hemato-biochemical analysis

The haemocyanin of haemolymph was estimated according to Chen and Cheng (1993a, b) and expressed as mmol/L. Serum total protein, glucose, total cholesterol and triacylglycerol were determined using respective kits procured from Erba^® Mannheim, Transasia Diagnostic Bio-Medicals Pvt. Ltd., Himachal Pradesh, India.

2.12 Statistical analysis

Prior to statistical analysis, all data were examined for homogeneity of variance to verify compliance with the assumptions of analysis of variance (ANOVA). Eack tank of different treatments was considered the experimental unit for statistical analysis, while individual shrimp sampled from each tank were treated as subsamples. Subsequently, statistical analyses were performed using IBM SPSS Statistics version 22.0 (IBM Corp., Armonk, NY, USA). Data generated from factorial experiments were analyzed by two-way ANOVA to evaluate the main effects of dietary protein and lipid, and LHA, as well as their interactions where applicable. Data from individual treatment comparisons were analyzed using one-way ANOVA. Results are presented as mean ± SEM for two-way ANOVA and mean ± SE for one-way ANOVA. Whenever one-way ANOVA indicated a significant treatment effect, Duncan’s multiple range test (DMRT) was used for post hoc pairwise comparisons among treatment means. Statistical significance was declared at 5% significance level (p<0.05).

3 Results

3.1 Proximate composition of experimental diets

Proximate composition viz. dry matter, CP, EE, CF, TA and NFE contents of experimental diets varied from 92.39 to 92.76, 36.38 to 30.05, 5.05 to 9.26, 4.21 to 4.82, 7.33 to 8.54, and NFE 45.34 to 49.05%, respectively (Table 1). The GE, DE and P: E were found to be in the ranges of 470.32-487.45 Kcal/100 g, 374.22-397.94 Kcal/100 g and 75.51-97.22 mg CP/Kcal DE, respectively. The results indicated the decreased CP and increased EE, GE and DE made the experimental hetero-nitrogenous, hetero-lipidic and hetero-caloric. Consequently, the high protein-low energy diets were converted to low protein-high energy diets with decreasing P: E value.

3.2 Water quality parameters

The water quality parameters did not vary significantly among the different experimental units (Table 2). The average water salinity, temperature, pH and osmolality of experimental units ranged between 10.33 ± 0.13 and 10.53 ± 0.08 g/L (p=0.9282), 26.81 ± 1.06 and 27.30 ± 1.36 °C (p=0.3202), 8.23 ± 0.13 and 8.44 ± 0.37 (p=0.3933) and 230.56 ± 3.06 and 238.28 ± 3.37 mosmol/kg (p=0.9766), respectively. However, the average water DO, total alkalinity (TA), total hardness (TH), ammonia nitrogen (NH₃-N), nitrite nitrogen (NO₂-N), nitrate nitrogen (NO₃-N), calcium ion (Ca²⁺), magnesium ion (Mg²⁺) and potassium ion (K⁺) of experimental units were found within the ranges of 7.07 ± 0.30 - 7.14 ± 0.09 (p=0.9475), 268.3 ± 1.76 - 273.33 ± 1.07 (p=0.1546), 2678.33 ± 4.31 - 2715.66 ± 4.20 (p=0.3232), 0.10 ± 0.01 - 0.14 ± 0.01 (p=0.1373), 0.011 ± 0.001 - 0.014 ± 0.001 (p=0.8294), 0.039 ± 0.001 - 0.048 ± 0.001 (p=0.4222), 335.3 ± 1.58 - 445.83 ± 3.07 (p=0.2103), 533.3 ± 6.58 - 543.33 ± 3.07 (p=0.9372) and 101.32 ± 3.63 - 103.3 ± 2.58 mg/L (p=0.1283), respectively. The free carbon dioxide was not detectable in water of any experimental unit.

Table 2. Water quality parameters of different experimental units during the experimental period of 60 days.

Data are expressed as mean ± SE, n=3 .

P₃₆L₅LHA₀, 360 g/kg crude protein, 50 g/kg lipid and 0 g/kg leonardite humic acid (LHA); P₃₆L₅LHA_0.9, P₃₆L₅LHA₀ diet supplemented with 9 g/kg LHA; P₃₆L₅LHA_1.2, P₃₆L₅LHA₀ diet supplemented with 12 g/kg LHA; P₃₃L₇LHA₀, 330 g/kg crude protein, 70 g/kg lipid and 0 g/kg LHA; P₃₃L₇LHA_0.9, P₃₃L₇LHA₀ diet supplemented with 9 g/kg LHA; P₃₃L₇LHA_1.2, P₃₃L₇LHA₀ diet supplemented with 12 g/kg LHA; P₃₀L₉LHA_0, 300 g/kg crude protein, 90 g/kg lipid and 0 g/kg LHA; P₃₀L₉LHA_0.9, P₃₀L₉LHA₀ diet supplemented with 9 g/kg LHA; P₃₀L₉LHA_1.2, P₃₀L₉LHA₀ diet supplemented with 12 g/kg LHA;

DO, dissolved oxygen;

CO₂, carbon dioxide;

NH₃-N, ammonia nitrogen;

NO₂-N, nitrite nitrogen;⁶NO₃-N, nitrate nitrogen;

Ca²⁺, calcium ion;

Mg²⁺, magnesium ion;

K⁺, potassium ion.

3.3 Growth, feed and nutrient utilization, body indices, and survival

There was a significant effect of varying dietary protein and lipid levels, a significant effect of LHA supplementation and a significant interaction between protein-lipid variations and LHA supplementation on the growth and nutrient utilization parameters and body indices of P. vannamei (Table 3). However, the survival rates were neither affected by the protein-lipid variation (p=0.0755) nor by the LHA levels (p=0.2014) and their interaction (p=0.0521). The growth parameters, WG (p=0.006), SGR (p=0.005), LER (p=0.003) and PER (p=0.007) were significantly higher in the P₃₃L₇ group compared to levels of protein and lipids. The lowest growth was observed at 0 g/kg LHA and the highest at 12 g/kg LHA supplemented group. The interaction revealed that LHA enhanced growth only under adequate lipid and protein levels, with maximum growth recorded in P₃₃L₇LHA_1.2 group and lowest in P₃₀L₉LHA₀ group. However, FCR and HPSI were significantly (p=0.0007, p=0.0005) lowest in P₃₃L₇ group compared to other dietary groups (Table 3). Highest and lowest HPSI (p=0.0004) and FCR (p=0.0003) were found in 0 g/kg LHA and 12 g/kg LHA fed group, respectively. There was significant interaction effect observed resulted in lowest (p=0.0003, p=0.0006) FCR and HPSI in shrimps of P₃₃L₇LHA_1.2 group while highest values observed in P₃₀L₉LHA₀ group.

Table 3. Growth, nutrient utilization, and body indices of juvenile Penaeus vannamei fed with leonardite humic acid supplemented with varying protein and lipid (energy) diets reared in inland saline water.

Data are expressed as mean ± SE, n=3 (One-way ANOVA) and mean and SEM, n=3 (Two-way ANOVA); Mean values in the same column with different superscripts differ significantly (p< 0.05);

P₃₆L₅LHA₀, 360 g/kg crude protein, 50 g/kg lipid and 0 g/kg leonardite humic acid (LHA); P₃₆L₅LHA_0.9, P₃₆L₅LHA₀ diet supplemented with 9 g/kg LHA; P₃₆L₅LHA_1.2, P₃₆L₅LHA₀ diet supplemented with 12 g/kg LHA; P₃₃L₇LHA₀, 330 g/kg crude protein, 70 g/kg lipid and 0 g/kg LHA; P₃₃L₇LHA_0.9, P₃₃L₇LHA₀ diet supplemented with 9 g/kg LHA; P₃₃L₇LHA_1.2, P₃₃L₇LHA₀ diet supplemented with 12 g/kg LHA; P₃₀L₉LHA_0, 300 g/kg crude protein, 90 g/kg lipid and 0 g/kg LHA; P₃₀L₉LHA_0.9, P₃₀L₉LHA₀ diet supplemented with 9 g/kg LHA; P₃₀L₉LHA_1.2, P₃₀L₉LHA₀ diet supplemented with 12 g/kg LHA.

WG, weight gain;

SGR, specific growth rate (%/day);

FCR, feed conversion ratio;

PER, protein efficiency ratio;

LER, lipid efficiency ratio;

HPSI, hepatopancreas somatic index.

3.4 Whole body proximate composition

Dietary protein and lipid levels had significant effect on whole-body proximate composition parameters, supplementing LHA had a significant effect only on whole body crude protein (p=0.0003) and crude lipid (p=0.0006) contents, and a significant interaction effect on crude protein (p=0.0003) and crude lipid (p=0.0008) contents (Table 4). Higher whole-body protein and lipid contents were found in P₃₃L₇ and P₃₀L₉ group, respectively, whereas, 12 g/kg LHA resulted in higher whole-body protein and lower crude lipid content. A significant interaction described the beneficial role of LHA with dietary protein-lipid levels resulted in highest and lowest whole-body protein content in P₃₃L₇LHA_1.2 and P₃₀L₉LHA₀ group, respectively, while lowest and highest lipid content in shrimps of P₃₃L₇LHA_1.2 and P₃₀L₉LHA₀ diet fed groups (p=0.0004, p=0.0002).

Table 4. Whole body proximate composition (% wet weight basis) of juvenile Penaeus vannamei fed with leonardite humic acid supplemented with varying protein and lipid diets reared in inland saline water.

Data are expressed as mean ± SE, n=3 (One-way ANOVA) and mean and SEM, n=3 (Two-way ANOVA); Mean values in the same column with different superscripts differ significantly (p< 0.05);

P₃₆L₅LHA₀, 360 g/kg crude protein, 50 g/kg lipid and 0 g/kg leonardite humic acid (LHA); P₃₆L₅LHA_0.9, P₃₆L₅LHA₀ diet supplemented with 9 g/kg LHA; P₃₆L₅LHA_1.2, P₃₆L₅LHA₀ diet supplemented with 12 g/kg LHA; P₃₃L₇LHA₀, 330 g/kg crude protein, 70 g/kg lipid and 0 g/kg LHA; P₃₃L₇LHA_0.9, P₃₃L₇LHA₀ diet supplemented with 9 g/kg LHA; P₃₃L₇LHA_1.2, P₃₃L₇LHA₀ diet supplemented with 12 g/kg LHA; P₃₀L₉LHA_0, 300 g/kg crude protein, 90 g/kg lipid and 0 g/kg LHA; P₃₀L₉LHA_0.9, P₃₀L₉LHA₀ diet supplemented with 9 g/kg LHA; P₃₀L₉LHA_1.2, P₃₀L₉LHA₀ diet supplemented with 12 g/kg LHA.

3.5 Activities of hepatopancreatic digestive and lipid metabolic enzymes

3.5.1 Hepatopancreatic digestive enzymes

Hepatopancreatic digestive enzyme amylase, protease and lipase activities of different dietary groups significantly affected by dietary lipid-protein levels (p=0.0004, p=0.0003, p=0.0004), LHA supplementation (p=0.0002, p=0.0002, p=0.0004), and interaction between dietary protein-lipid levels and LHA inclusion of white-leg shrimp, P. vannamei juveniles (Table 5).

Table 5. Digestive enzyme activities of juvenile Penaeus vannamei fed with leonardite humic acid supplemented with varying protein and lipid diets reared in inland saline water.

Data are expressed as mean ± SE, n=3 (One-way ANOVA) and mean and SEM, n=3 (Two-way ANOVA); Mean values in the same column with different superscripts differ significantly (p< 0.05);

P₃₆L₅LHA₀, 360 g/kg crude protein, 50 g/kg lipid and 0 g/kg leonardite humic acid (LHA); P₃₆L₅LHA_0.9, P₃₆L₅LHA₀ diet supplemented with 9 g/kg LHA; P₃₆L₅LHA_1.2, P₃₆L₅LHA₀ diet supplemented with 12 g/kg LHA; P₃₃L₇LHA₀, 330 g/kg crude protein, 70 g/kg lipid and 0 g/kg LHA; P₃₃L₇LHA_0.9, P₃₃L₇LHA₀ diet supplemented with 9 g/kg LHA; P₃₃L₇LHA_1.2, P₃₃L₇LHA₀ diet supplemented with 12 g/kg LHA; P₃₀L₉LHA_0, 300 g/kg crude protein, 90 g/kg lipid and 0 g/kg LHA; P₃₀L₉LHA_0.9, P₃₀L₉LHA₀ diet supplemented with 9 g/kg LHA; P₃₀L₉LHA_1.2, P₃₀L₉LHA₀ diet supplemented with 12 g/kg LHA;

Amylase activity is expressed in micromole of maltose released/mg protein/min; ³Protease activity is expressed in millimole of tyrosine released/mg protein/min; ⁴Lipase activity is expressed in unit/mg protein/h.

The digestive enzyme activities, amylase and lipase were significantly (p=0.0003, p=0.0004) higher in P₃₀L₉ group and lowest in observed in P₃₆L₅ group, whereas, higher and lower protease activity was found in P₃₃L₇ and P₃₀L₉ group, respectively. The highest and lowest digestive enzyme activities were at 12 g/kg LHA and 0 g/kg LHA supplemented group, respectively. There was a significant interaction effect observed on hepatopancreatic protease and lipase activities showing the promising effect of LHA with sufficient dietary protein-lipid levels resulted in maximal enzymatic activities in shrimps of P₃₃L₇LHA_1.2 group (p=0.0003, p=0.0002, p=0.0003), while lowest amylase activities found in P₃₆L₅LHA₀ group, protease activities in P₃₀L₉LHA₀ and P₃₀L₉LHA_0.9 groups, lipase activities in P₃₆L₅LHA₀ and P₃₆L₅LHA_0.9 groups.

3.5.2 Hepatopancreatic lipid metabolic enzymes

There was a significant effect of varying dietary protein and lipid levels (p=0.0003, p=0.0005), a significant effect of LHA supplementation (p=0.0002, p=0.0002) and a significant interaction between protein-lipid variations and LHA supplementation (p=0.0003, p=0.0004) on the lipid metabolic enzymes G6PDH and CPT-1 of P. vannamei (Table 6). The activities of G₆PDH and CPT-1 were significantly higher in the P₃₃L₇ group, whereas, lowest G₆PDH and CPT-1 activities recorded in P₃₆L₅ group compared to other protein-lipid levels. The lowest activities was observed at 0 g/kg LHA and the highest at 12 g/kg LHA fed group. Dietary protein-lipid levels and LHA showed a significant interaction effect depicting the lipid utilization capacity of LHA in shrimp by elevating the activities of these enzymes in response to higher doses of LHA and highest recorded in P₃₃L₇LHA_1.2 diet fed group (p=0.0004, p=0.0006), while shrimps fed without LHA in protein-lipid combinations showed lower activities of these enzymes and lowest recorded in P₃₆L₅LHA₀ group.

Table 6. Lipid metabolic enzyme activities of juvenile Penaeus vannamei fed with leonardite humic acid supplemented with varying protein and lipid diets reared in inland saline water.

Data are expressed as mean ± SE, n=3 (One-way ANOVA) and mean and SEM, n=3 (Two-way ANOVA); Mean values in the same column with different superscripts differ significantly (p< 0.05);

P₃₆L₅LHA₀, 360 g/kg crude protein, 50 g/kg lipid and 0 g/kg leonardite humic acid (LHA); P₃₆L₅LHA_0.9, P₃₆L₅LHA₀ diet supplemented with 9 g/kg LHA; P₃₆L₅LHA_1.2, P₃₆L₅LHA₀ diet supplemented with 12 g/kg LHA; P₃₃L₇LHA₀, 330 g/kg crude protein, 70 g/kg lipid and 0 g/kg LHA; P₃₃L₇LHA_0.9, P₃₃L₇LHA₀ diet supplemented with 9 g/kg LHA; P₃₃L₇LHA_1.2, P₃₃L₇LHA₀ diet supplemented with 12 g/kg LHA; P₃₀L₉LHA_0, 300 g/kg crude protein, 90 g/kg lipid and 0 g/kg LHA; P₃₀L₉LHA_0.9, P₃₀L₉LHA₀ diet supplemented with 9 g/kg LHA; P₃₀L₉LHA_1.2, P₃₀L₉LHA₀ diet supplemented with 12 g/kg LHA;

G₆PDH, glucose-6-phosphate dehydrogenase, the activity is expressed as mM phosphorus released/min/mg protein; ³CPT-I, carnitine palmitoyltransferase I, the activity is expressed as mM DTNB converted/min/mg protein.

3.6 Oxidative stress enzyme activities and malondialdehyde levels

Hepatopancreatic and branchial SOD and CAT activities and hepatopancreatic MDA levels were significantly affected by dietary protein-lipid levels, LHA levels and their interaction in P. vannamei juveniles reared in ISW (Table 7). Significantly lowest and highest SOD (p=0.0003, p=0.0003) and CAT (p=0.0004, p=0.0002) activities and MDA (p=0.0003) content were found in P₃₃L₇ and P₃₀L₉ group, respectively. LHA supplementation decreased SOD (p=0.0004, p=0.0002) and CAT (p=0.0003, p=0.0005) activities and MDA level (p=0.0005) and feeding at 12 g/kg showed the lowest SOD and CAT activities and MDA in shrimps. Dietary protein-lipid and LHA had a significant interaction effect on SOD (p=0.0004, p=0.0003) and CAT activities (p=0.0004, p=0.0002) and MDA content (p=0.0004) deciphering the role of LHA with optimized protein-lipid level, resulted in lowest antioxidant enzymes activities and MDA content (p=0.0003) in P₃₃L₇LHA_1.2 group. However, diets without LHA supplementation showed higher activities of SOD and CAT and MDA level and highest values found in highest lipid fed group with no LHA i.e. P₃₀L₉LHA₀ group.

Table 7. Oxidative stress enzyme activities and MDA level of juvenile Penaeus vannamei fed with leonardite humic acid supplemented with varying protein and lipid diets reared in inland saline water.

Data are expressed as mean ± SE, n=3 (One-way ANOVA) and mean and SEM, n=3 (Two-way ANOVA); Mean values in the same column with different superscripts differ significantly (p< 0.05);

P₃₆L₅LHA₀, 360 g/kg crude protein, 50 g/kg lipid and 0 g/kg leonardite humic acid (LHA); P₃₆L₅LHA_0.9, P₃₆L₅LHA₀ diet supplemented with 9 g/kg LHA; P₃₆L₅LHA_1.2, P₃₆L₅LHA₀ diet supplemented with 12 g/kg LHA; P₃₃L₇LHA₀, 330 g/kg crude protein, 70 g/kg lipid and 0 g/kg LHA; P₃₃L₇LHA_0.9, P₃₃L₇LHA₀ diet supplemented with 9 g/kg LHA; P₃₃L₇LHA_1.2, P₃₃L₇LHA₀ diet supplemented with 12 g/kg LHA; P₃₀L₉LHA_0, 300 g/kg crude protein, 90 g/kg lipid and 0 g/kg LHA; P₃₀L₉LHA_0.9, P₃₀L₉LHA₀ diet supplemented with 9 g/kg LHA; P₃₀L₉LHA_1.2, P₃₀L₉LHA₀ diet supplemented with 12 g/kg LHA.

CAT, catalase, the activity is expressed as nanomoles of H₂O₂ decomposed/mg protein/min;

SOD, superoxide dismutase, the activity is expressed as 50% inhibition of epinephrine autoxidation/mg protein/min;

MDA, malondialdehyde, the level is expressed in nanomole/mg protein.

3.7 Haemocyanin concentration and serum biochemical profile

Varying dietary protein-lipid levels, levels of LHA and interaction between protein-lipids and LHA had a significant effect on haemocyanin and serum total protein, glucose, cholesterol and triacylglycerol (TAG) contents in P. vannamei juveniles reared in ISW (Table 8). Significantly highest and lowest haemocyanin (p=0.0002) and serum total protein (p=0.0005) content was recorded in shrimps of P₃₃L₇ group and P₃₀L₉ group, respectively. However, glucose (p=0.0003), cholesterol (p=0.0005) and TAG (p=0.0006) contents increased significantly with decreasing dietary protein and increasing lipid levels and highest found in P₃₀L₉ group. Dietary 12 g/kg LHA level showed highest haemocyanin (p=0.0004) and total protein (p=0.0005) contents but lowest glucose (p=0.0003), cholesterol (p=0.0002) and TAG (p=0.0003) contents. A positive interaction effect was observed except hemocyanin content. Dietary supplementation of LHA in protein-lipid levels significantly increased haemocyanin and total protein contents in shrimps than non-supplemented counterparts and highest recorded in shrimps of P₃₃L₇LHA_1.2 group. However, an opposite trend was observed in serum glucose, cholesterol and TAG contents fed with LHA and highest and lowest values observed in P₃₀L₉LHA₀ and P₃₃L₇LHA_1.2 groups, respectively.

Table 8. Hemocyanin and serum biochemical profile of juvenile Penaeus vannamei fed with leonardite humic acid supplemented with varying protein and lipid diets reared in inland saline water.

Data are expressed as mean ± SE, n=3 (One-way ANOVA) and mean and SEM, n=3 (Two-way ANOVA); Mean values in the same column with different superscripts differ significantly (p< 0.05).

P₃₆L₅LHA₀, 360 g/kg crude protein, 50 g/kg lipid and 0 g/kg leonardite humic acid (LHA); P₃₆L₅LHA_0.9, P₃₆L₅LHA₀ diet supplemented with 9 g/kg LHA; P₃₆L₅LHA_1.2, P₃₆L₅LHA₀ diet supplemented with 12 g/kg LHA; P₃₃L₇LHA₀, 330 g/kg crude protein, 70 g/kg lipid and 0 g/kg LHA; P₃₃L₇LHA_0.9, P₃₃L₇LHA₀ diet supplemented with 9 g/kg LHA; P₃₃L₇LHA_1.2, P₃₃L₇LHA₀ diet supplemented with 12 g/kg LHA; P₃₀L₉LHA_0, 300 g/kg crude protein, 90 g/kg lipid and 0 g/kg LHA; P₃₀L₉LHA_0.9, P₃₀L₉LHA₀ diet supplemented with 9 g/kg LHA; P₃₀L₉LHA_1.2, P₃₀L₉LHA₀ diet supplemented with 12 g/kg LHA;

TAG, triacylglycerol.

4 Discussion

Aquaculture production is projected to require a 96% increase over present levels by 2050 to meet the anticipated demand for animal protein in human diets (FAO, 2022). This huge production is possible through implementation of feed-based intensified aquaculture with appropriate nutritional strategies to make the fish culture sustainable as feed is the main contributor to the ecological footprint. Protein is the costliest nutrient in aquafeed. Thus, more dietary protein with inadequate non-protein energy source (lipid and carbohydrate) not only enhances the feed cost also causes environmental pollution through more excretion of ammonia with reduced production and making the aquaculture operation un-sustainable (Jana et al., 2021a; Talukdar et al., 2021). In this regard, use of low protein and high lipid (energy) aquafeed with appropriate protein-to-energy ratio probably could ensure eco-friendly and sustainable aquaculture production. Moreover, supplementing diet with either lipid utilizing or high dietary lipid mediated harmful effect mitigating nutraceuticals probably could optimize the fish/shrimp production. In this direction, LHA could be one of the important and practical choices as it is an antioxidant with having lipo-metabolic properties (Rath et al., 2006; Li et al., 2016).

Water temperature, pH and DO were found to be within the ideal ranges of white-leg shrimp, P. vannamei culture (Talukdar et al., 2021; Chuphal et al., 2021). Salinity is a critical water quality parameter of ISW. The 10 ppt water salinity of the current study corroborated the reported value of Talukdar et al. (2021) for improved growth and survival of P. vannamei juveniles. The optimum level of water total alkalinity and total hardness play a major role in the molting and maintaining the biological processes of shrimp (Boyd and Tucker, 1998). Accordingly, the total hardness and total alkalinity of the current study were within the ideal ranges for vannamei culture in ISW. In the present study, the NH₃-N, NO₂-N and NO₃-N, K⁺, Ca²⁺, and Mg²⁺ concentration of ISW were within the permissible ranges for vannamei culture (Reham et al., 2019; Jana et al., 2021a). The ions (calcium, sodium, and potassium) play a crucial role in the osmoregulatory process and maintaining the membrane potentials (Panmei et al., 2023; Jaffer et al., 2024). Shrimp require minerals for metabolism of nutrients (metabolism of lipids, proteins and carbohydrates) and growth where soluble minerals are constituents of tissues and act as enzymatic cofactors (Panmei et al., 2023). Among the water ions, potassium is considered a limiting element for shrimp including P. vannamei culture in ISW. As deficient, the potassium fortification of ISW is recommended for a successful shrimp culture (Lakra et al., 2014; Jana et al., 2021a). Water osmolality in this study was within the desirable limit for shrimp culture to corroborate the findings of Talukdar et al. (2021).

Growth occurs after nutrients accumulation in the body as a resultant consequence of their metabolic action viz., anabolism and catabolism. Among different nutrients, dietary protein has the major role for growth performance of animals including shrimp provided non-protein energy producing nutrients are available adequately with optimum protein to energy (P: E) value (Roy et al., 2007a). This may be attributed to the protein-sparing effect of lipids, where dietary lipid or carbohydrate to fulfil the energy demand and dietary protein probably can be directed towards the growth of the animals (Hu et al., 2008). Accordingly, in the present study, the highest WG, SGR, and PER with the lowest FCR was found in shrimps of 330 g/kg CP and 70 g/kg lipid (P₃₃L₇) fed group. However, further decreasing dietary protein and increasing lipid level, resulted in growth retardation of shrimp indicates that excess dietary lipid caused metabolic burden and could not be efficiently utilized by shrimp as their limited potential to synthesize bile acid. Moreover, dietary supplementation of nutraceuticals probably can further improve the growth performance of animals. In this regard, dietary microbial humic acid can be considered as one of important growth enhancers for animals (Rasidi et al., 2021). Additionally, its colloidal characteristics and high capacity to form aggregates within solutions creates a protective barrier on the epithelium membrane of the gastrointestinal tract against infections and toxins (Van der Klis et al., 1993). It also increases the integrity of intestinal villi with a slower rate of feed passage through the gut (Van der Klis et al., 1993) leading to enhanced exposure time of nutrients to digestive enzymes ensuring effective utilization of feed and nutrients in animals (Ji et al., 2006; Taklimi et al., 2012). Thus, in the current study, 12 g/kg dietary supplementation of LHA improved the growth performance of shrimp and overall, the highest WG, SGR, PER and LER with the lowest FCR was found in shrimps of 330 g/kg CP, 70 g/kg lipid and 12 g/kg LHA fed group (P₃₃L₇LHA_1.2) showing its potential to utilize high dietary lipid level in shrimp through slow feed passage in gut, thus enhances nutrient exposure to intestinal digestive enzymes and improves feed utilization efficiency. Similarly, Rousdy and Wijayanti (2015) and Gao et al. (2018) reported the best growth performance, respectively, in Cyprinus carpio fed with 10 g/kg humic acid and L. vannamei fed with 12 g/kg fulvic acid supplemented diets. Moreover, to corroborate our findings, previous studies demonstrated that dietary humic acid significantly increased growth performance of juvenile loach (Paramisgurnus dabryanus) (Gao et al., 2017), Nile tilapia (Jusadi et al., 2020) and rainbow trout (Oncorhynchus mykiss) (Lieke et al., 2021).

Hepatopancreas, the largest organ in shrimp, plays a central role in the digestive enzyme synthesis and secretion, digestion, and absorption of nutrients (Wang et al., 2014; Xu et al., 2016). The size of HPSI represents the physiological and metabolic status of shrimp and larger size indicates fatty liver with excessive fat deposition (Xie et al., 2019). Thus, in the present study, the highest HPSI value 300 g/kg CP and 90 g/kg lipid fed groups might indicate the more fat deposition in the hepatopancreas of shrimp. However, the shrimps of 330 g/kg CP and 70 g/kg lipid (P₃₃L₇) fed group in this study showed the lowest HPSI value. This finding corroborated the observation of González-Félix et al. (2002); Xie et al. (2019) and Chuphal et al. (2021) who concluded that HPSI value of shrimp could increases with the increasing dietary lipid level. However, dietary supplementation of 12 g/kg LHA in the present study significantly decreased the HPSI value with overall the lowest value in shrimps of 330 g/kg CP, 70 g/kg lipid and 12 g/kg LHA (P₃₃L₇LHA_1.2) fed group. LHA is known to upregulate CPT-1 for promoting fatty acid transport and β-oxidation, enhancing lipid utilization and energy production. In corroboration with the present finding, Deng et al. (2020) reported the reduced hepatosomatic index (HSI) value in GIFT tilapia (Oreochromis niloticus) due to feeding of LHA. However, in contrast, Prokešová et al. (2021) did not find any significant variation of HPSI value in African Catfish (Clarias gariepinus) due to feeding of LHA. Insignificant variation of survival rate among different dietary groups of shrimps fed with varying levels of protein, lipid and LHA nutraceuticals could corroborate the earlier findings in P. vannamei (Zhou et al., 2017; Yilmaz et al., 2018). Moreover, Rath et al. (2006) suggested that dietary humic acid could exert a negative effect on the growth performance of broiler chicken. However, in contrast, Aiken et al. (1986) found increased survival of fish due to dietary humic acid through enhanced immunity. The inconsistent results observed in different studies might be due to variation of dietary protein and lipid levels, dose of LHA, species and age of animals. Overall, 330 g/kg CP, 70 g/kg lipid and 12 g/kg LHA (P₃₃L₇LHA_1.2) in the diet with P: E value of 86.44 mg CP/Kcal DE could be most favorable to maximize growth and nutrient utilization of white-leg shrimp, P. vannamei reared in ISW of 10 ppt.

Whole-body proximate composition of shrimp indicates their nutrition status (Bhusare et al., 2023). In the current study, decreasing dietary protein and increasing dietary lipid levels significantly decreased the whole-body lipid content of shrimps with the highest in 300 g/kg CP and 90 g/kg lipid fed group. This trend might be due high dietary lipid mediated more fat deposition in the hepatopancreas with enhanced HPSI value of shrimp (Xu et al., 2018). On the other hand, lower whole body lipid content in lower lipid fed groups probably could indicate the optimum utilization of dietary lipid for energy production (Zhang et al., 2013; Chuphal et al., 2021). Similar observations were also previously reported in P. vannamei (Zhu et al., 2010; Jannathulla et al., 2019). However, in our study, the highest whole body protein content was found in the shrimp of 330 g/kg CP and 70 g/kg lipid (P₃₃L₇) fed group due to protein sparing effect of dietary lipid with higher protein accretion and retention. On the other hand, 12 g/kg dietary LHA significantly enhanced and decreased the whole-body protein and lipid content, respectively with the highest protein and lower lipid level in P₃₃L₇LHA_1.2 fed group. However, this group showed similar whole body lipid content to 360 g/kg CP, 50 g/kg lipid and 9 g/kg LHA (P₃₆L₅LHA_0.9) and 360 g/kg CP, 50 g/kg lipid and 12 g/kg LHA (P₃₆L₅LHA_1.2) fed groups. Similar results were also reported in earlier studies (Wu et al., 2009; Vucskits et al., 2010; Kunavue and Lien, 2012). However, in contrast to our observation, Zhu et al. (2010) and Wang et al. (2014) concluded that feeding of LHA with varying levels of dietary lipids could not produce any significant effect on the whole-body protein content of shrimp. These inconsistent findings might be due to variation of dietary levels of humic acid, time of conducting experiment, species etc.

Nutrient digestibility of shrimp depends on hepatopancreatic digestive enzyme activities and dietary inclusion of additives may stimulate their synthesis and secretion for improving nutrient digestibility and growth performance (Chuphal et al., 2021). Further, the activities of digestive enzymes depend on the availability of enzyme-specific substrates in the hepatopancreas of shrimp (Talukdar et al., 2021; Jana et al., 2022). Accordingly, in the present study, the amylase and lipase activities significantly increased with increasing dietary lipid level. However, the highest and the lowest protease activities were found respectively in 330 g/kg CP and 70 g/kg lipid (P₃₃L₇) and 300 g/kg protein and 90 g/kg lipid (P₃₀L₉) fed groups. Similarly, feeding of high dietary lipid significantly increased hepatopancreatic lipase activity in shrimp (Chuphal et al., 2021) and decreased intestinal protease activity in GIFT tilapia (Bhusare et al., 2023). On the other hand, in the current study, 12 g/kg dietary LHA significantly enhanced the hepatopancreatic protease, lipase and amylase activities of shrimp with overall the highest activities in 330 g/kg protein, 70 g/kg lipid and 12 g/kg LHA (P₃₃L₇LHA_1.2) fed group leading to improved growth performance probably mediated through improved nutrient digestibility and utilization (Windisch et al., 2008; Taklimi et al., 2012). In the line of present result, 6 g/kg dietary humic acid sodium salt could enhance the activities of pepsin, amylase, trypsin, and lipase in fish (Yilmaz et al., 2018) with up-regulated expressions (Zhang et al., 2018; Mao, 2019; Dou and Wu, 2023). Similarly, Gao et al. (2017) reported that 15-20 g/kg dietary fulvic acid could enhance protease and lipase activities but did not affect α-amylase activity in juvenile loach. Gao et al. (2018) also reported that 12 g/kg dietary fulvic acid could enhance the activities of protease, lipase and amylase in L. vannamei.

CPT-1 enzyme helps in transporting long chain fatty acids (LCFAs) from cytoplasm to mitochondria where the fatty acids undergo ß-oxidation to generate energy (Raghuvaran et al., 2023). Thus, the activity of this enzyme indicates the status of lipid metabolism in fish and shellfish. Accordingly, in our study, the highest hepatopancreatic CPT-I activity in the shrimp of 330 g/kg protein, 70 g/kg lipid and 12 g/kg LHA (P₃₃L₇LHA_1.2) fed groups probably could accelerate ß-oxidation of fatty acids leading to reduced body lipid accumulation. In agreement with the present finding, Lu et al. (1994) found that dietary humic acid metal complex significantly increased hepatic CPT-1 activity in fish with subsequent fatty acid oxidation resulting in a reduction of body lipid accumulation. The G₆PDH enzyme indirectly plays an important role in the lipid anabolism through producing and supplying NADPH via pentose phosphate pathway for fatty acid synthesis leading to improved growth of animals including fish (Verri et al., 2001). LHA has been shown to stimulate key metabolic enzymes in fish, notably CPT-1 and G₆PDH. Upregulation of CPT-1 enhances fatty acid transport and β-oxidation, thereby improving lipid utilization and energy production. Simultaneously, increased G6PDH activity elevates NADPH generation, which is crucial for antioxidant defense and various biosynthetic pathways. This dual modulation of metabolic enzymes may underlie the observed improvements in growth performance, metabolic efficiency, and stress tolerance in fish. In the current study, the shrimp P₃₃L₇LHA_0.12 fed group showed the highest G₆PDH activity with improved growth. Similar results obtained by Sun et al. (2018) and Sun et al. (2020) who demonstrated that higher G₆PDH activity could be related to enhanced growth and improved physiological responses of fish.

Oxidative stress in fish and mammalian cells is a result of the excess production of reactive oxygen species (ROS), which induces severe oxidative damage resulting in cellular death (Karmakar et al., 2021). Shrimps has own antioxidant defense mechanisms where cytosolic SOD is the first and most crucial defense line that eliminates the excess of superoxide anion (O₂^-) by converting it into comparatively less harmful hydrogen peroxide (H₂O₂), which is then catalyzed by CAT to harmless O₂ and H₂O (Matozzo et al., 2011) and thus a dynamic balance between free radical generation and elimination from shrimp body is maintained (Atli and Canli, 2010). Thus, enhanced activities of SOD and CAT indicate the stress in shrimp (Matozzo et al., 2011). Accordingly, in the present study, the enhanced SOD and CAT activities in shrimps of high lipid (90 g/kg) and low protein (300 g/kg) fed group probably could indicate the high lipid mediated metabolic stress (Zhang et al., 2013; Xu et al., 2018; Chuphal et al., 2021; Raghuvaran et al., 2023). However, 12 g/kg dietary LHA in this study probably could reduce stress through scavenging ROS with resultant reduced activities of the SOD and CAT and consequently the lowest activities of these enzymes were found in the shrimps of 330 g/kg protein, 70 g/kg lipid and 12 g/kg LHA (P₃₃L₇LHA_1.2) fed group (Tan et al., 2017; Lieke et al., 2021). Phenolic and carboxylic groups present in LHA also provide strong antioxidant effects by scavenging free radicals and reducing oxidative stress. Earlier studies also reported the similar finding in Chinese shrimp, P. chinensis (Zhao et al., 2001), catfish, Clarias gariepinus (Prokešová et al., 2021), ayu fish, Plecoglossus altivelis (Nakagawa et al., 2009), common carp, C. carpio (Abdel-Wahab et al., 2012) and L. vannamei (Gao et al., 2021) due to feeding of dietary LHA.

MDA is a harmful end product of poly unsaturated fatty acids (PUFAs) oxidation in the cells of animals including fish and shellfish (Bhusare et al., 2023). High cellular MDA level leads to cytotoxicity and cell damage of shrimp (Li et al., 2010). Accordingly, in the present study, increasing the dietary lipid level significantly increased the hepatopancreatic MDA level might be due to higher lipid mediated metabolic stress in shrimp. Similar observation was also previously reported in P. vannamei (Zhang et al., 2013). On the other hand, in our study, dietary LHA significantly decreased hepatopancreatic MDA levels with the most pronounced reduction in shrimp of P₃₃L₇LHA_1.2 fed group. In accordance with the present finding, Hudák et al. (2021) reported the reduced hepatopancreatic MDA content in 7 g/kg LHA fed shrimp.

Haemocyanin concentration in haemolymph is the indicator for health status of shrimp. This molecule has a secondary role in storing amino acids and serving as a source of free amino acids (FAA) needed for growth, energy production, immune function, and osmoregulation (Cuzon et al., 2004). The hemocyanin constitutes about 60-97% of the total hemolymph protein (Pascual et al., 2003). In our study, the lowest hemocyanin concentration was found in the shrimp of 300 g/kg protein and 90 g/kg (P₃₀L₉) lipid fed group might be due to less availability of dietary protein derived amino acids for synthesis of haemocyanin in the body (Talukdar et al., 2021). On the other hand, dietary supplementation of LHA enhanced the haemocyanin concentration with the highest concentration in the shrimp of P₃₃L₇LHA_1.2 fed group (Cuzon et al., 2004; Hamidoghli et al., 2020). To the best of our knowledge, there is no literature to support our findings on dietary LHA induced hemocyanin concentration of shrimp. However, Raghuvaran et al. (2023) found that the P. vannamei fed with 330 g/kg protein, 80 g/kg lipid and 0.5 g/kg L-carnitine had the highest hemocyanin concentration.

Serum biochemical parameters are good indicators of the nutritional, physiological and health status of animals. In the present study, the shrimp fed with 300 g/kg protein and 90 g/kg lipid (P₃₀L₉) showed the lowest serum total protein, whereas the highest value was found in 330 g/kg protein and 70 g/kg lipid (P₃₃L₇) fed group. Similar to our observation, Wang et al. (2014) reported that the serum total protein content of L. vannamei decreased with increasing dietary lipid levels. However, in contrast, Jannathulla et al. (2019) found that serum total protein increased with increasing dietary lipid of shrimp. In the present study, dietary LHA significantly enhanced serum total protein content with overall the highest value in the shrimp of 330 g/kg protein, 70 g/kg lipid and 12 g/kg LHA (P₃₃L₇LHA_1.2) fed group. To support the result of our study, earlier studies reported the significantly improved serum total protein content of fish due to feeding with LHA supplemented diets (Zhorina and Stepchenko, 1993; Maysa and Sheikh, 2008; Písaříková et al., 2010; Yalcin et al., 2006; Dell’Anno et al., 2020).

Serum glucose is considered as a stress marker and its concentration is positively correlated to stress in fish (Prokešová et al., 2021). Both dietary and mobilized glucose reflect the serum concentration (Jaffer et al., 2024). In our study, serum glucose concentration significantly increased with increasing dietary lipid and decreasing protein levels with the highest and the lowest values in shrimp of 300 g/kg protein and 90 g/kg lipid (P₃₀L₉) and 360 g/kg protein and 50 g/kg lipid (P₃₆L₅) fed groups, respectively. The highest serum glucose level might be due to high level of carbohydrates in the diet. Moreover, in order to fulfil the energy demand, the high dietary lipid induced stressed shrimp probably could mobilize glucose through glycogenolytic pathway leading to enhanced serum glucose concentration. Similarly, results were also reported in earlier studies (Wang et al., 2015; Talukdar et al., 2021). However, in contrast, increasing dietary lipid levels had no influence on serum glucose concentration in P. vannamei (Xu et al., 2018; Hamidoghli et al., 2020). On the other hand, in our study, dietary LHA significantly reduced serum glucose level probably through enhancing dietary carbohydrate metabolism and mitigating high dietary lipid mediated harmful effects. Overall, the lowest serum glucose concentration was found in shrimps of P₃₃L₇LHA_0.12 fed group. Similarly, Banaszkiewicz and Drobnik (1994) reported the dietary LHA induced reduced serum glucose level in fish.

Serum cholesterol and TAG levels are major indicators of nutritional and health status of fish including shrimp (Maheswaran et al., 2008; Talukdar et al., 2021). Cholesterol is the major constituent of the cell membrane, precursor of bile acid and steroid hormones and regulates the molt cycle of shrimp (Xie et al., 2019). Whereas, TAG play an important role in primary metabolic reserve and production of energy (Ciaramella et al., 2014). However, high serum concentration of both cholesterol and TAG is considered the poor health condition of shrimp. In our study, serum cholesterol and TAG level increased significantly with the increasing lipid and decreasing protein level of the diet might be due to the reason that the enhanced dietary lipid level probably could decrease the metabolic utilization of cholesterol and TAG at the tissue level (Zhang et al., 2013; Xu et al., 2016). However, dietary LHA significantly decreased serum cholesterol and TAG levels with overall the lowest concentrations in shrimps of P₃₃L₇LHA_1.2 fed group probably through enhancing lipid metabolism (Young and Foegeding, 1993; Abdo and Zeinb, 2004). Similar results were also reported in juvenile largemouth bass, Micropterus salmoides (Chen et al., 2020) and grey mullet, Mugil cephalus (Akbary et al., 2019). However, in contrast, Hakan et al. (2012) and Ozturk et al. (2012) could not find any effect of dietary humate on lipid metabolism of animals.

5 Conclusion

Feeding 12 g/kg leonardite humic acid (LHA) with 330 g/kg CP and 70 g/kg lipid (P: E 86.44 mg CP/Kcal DE) can improve the growth, nutrient utilization and antioxidant status of juvenile white shrimp, P. vannamei reared in ISW of 10 ppt salinity. However, further research is recommended for optimizing the inclusion level of leonardite humic acid in diet of shrimp.

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Footnotes

Edited by: Cosmas Nathanailides, University of Ioannina, Greece

Reviewed by: Amel Mohamed El Asely, Benha University, Egypt

Khanh Q. Nguyen, Auburn University, United States