Effect of Selected Nutraceuticals on Growth and Metabolism of Penaeus vannamei Juveniles Reared in Inland Saline Water

The global population has been growing at a steady rate of 1.05% per year in the last decade and it has created tremendous pressure on all food-producing sectors to meet global demands. Aquaculture has been the fastest-growing animal food-producing sector, contributing about 20% of animal protein needs worldwide (Raghuvaran et al., 2024).  However, the stagnation in capture fisheries has increased the demand for aquaculture, as it must produce an additional 47.5 million tonnes of fish by 2050. This high demand can be fulfilled only by the expansion of aquaculture resources. However, the non-availability of sufficient land and water sources has made vertical expansion impossible and hence, the horizontal expansion of available resources is the only viable option left. One such option with potential is using salt-affected inland areas for aquaculture purposes to enhance food security. These saline lands are unproductive and unsuitable for other agricultural activities (Jana et al., 2021). Using these wastelands not only improves food security but also generates additional income. Hence, the culture of euryhaline fish species in ISW has become popular among farmers globally (Roy et al., 2010). Among the crustaceans, Penaeus vannamei has become a candidate species for aquaculture due to its faster growth rate and wide tolerance to salinity (Chuphal et al., 2021; Raghuvaran et al., 2023). However, the imbalance of ions in ISW affects growth and immunity and increases the energy demands of the cultured species (Singha et al., 2021; Thirunavukkarasar et al., 2022).
       
Feed alone contributes 50-60% of the total operational expenses of aquaculture (Raghuvaran et al., 2024; Dheeran et al., 2025). Among dietary macronutrients, protein is the costliest and is vital in fish growth. The protein and lipid requirement of P. vannamei in ISW is 35.6% (Nanda, 2019) and 5.13% (Chuphal et al., 2021), respectively, at 10 ppt ambient salinity. This higher protein demand increases the production cost and is detrimental to sensitive culture systems like ISW, as this increases the release of nitrogenous wastes into the system. Hence, using low protein high energy diets is a viable alternative option in ISW. The protein-sparing effect of lipids can be better utilized by increasing the dietary lipid content in fish feed. But, the inclusion of high lipids in the diet results in excessive fat deposition in the body tissues and ultimately affects the health of the cultured organisms (Bhusare et al., 2023; Mai et al., 2025). In this regard, earlier studies conducted found that lipid-utilizing nutraceuticals such as L-carnitine (Raghuvaran et al., 2023), Taurine (Bhavatharaniya, 2021) and Quercetin (Unpublished data) have been found to improve growth and lipid utilization in P. vannamei reared in ISW. However, the information on the combinatorial effect of these nutraceuticals in P. vannamei is still lacking. Implementing the ideal protein concept has been found to reduce protein requirement from 35.16 to 32.09% without compromising the growth of P. vannamei reared in ISW (Maiti, 2022). Hence, this study was conducted to evaluate the effect of selected nutraceuticals (L-carnitine, Taurine and Quercetin) and limiting amino acids (Lysine and Methionine) on the growth and metabolic responses of P. vannamei reared in ISW of 10 ppt salinity.

Ethical statement
 
The ethical committee of ICAR-Central Institute of Fisheries Education, Mumbai, India, approved this study.
 
Formulation and preparation of experimental diets
 
Eight isonitrogenous (32% CP), isolipidic (6%) and isocaloric (384 Kcal digestible energy, DE/100 g) practical diets were supplemented with three different nutraceuticals (L-carnitine, taurine and quercetin) to formulate (Table 1) and prepare eight experimental diets, viz. Control, optimum dietary protein (32%) and lipid level (6%) with limiting amino acids and no supplementation of nutraceuticals; TC, Control diet supplemented with 0.05% L-carnitine; TT, Control diet supplemented with 0.5% Taurine; TQ, Control diet supplemented with 0.04% Quercetin; TC+Q, Control diet supplemented with L-Carnitine (50% dose) + Quercetin (50% dose); TC+T, Control diet supplemented with L-Carnitine (50% dose) + Taurine (50% dose); TT+Q, Control diet supplemented with Quercetin (50% dose) + Taurine (50% dose); TC+T+Q, Control diet supplemented with L-carnitine +Taurine+ Quercetin (33.3% dose each). All ground ingredients, except for feed additives, BHT and oils, were uniformly mixed and water was added as needed to form a dough. This dough was placed in a heat-resistant plastic bag and steam-cooked for 25 minutes before being cooled. The remaining ingredients were then thoroughly blended into the dough. Subsequently, the dough was pressed through a pelletizer (Uniextrude-single screw extruder; S. B. Panchal and Co. Mumbai, India) fitted with a perforated die of 1 mm diameter to make the pellets of the same diameter and initially air-dried at room temperature, then dried at 40^oC until the moisture content was reduced to approximately 10%. The dried pellets were then crushed into smaller sizes and stored at 4oC until use for feeding.


 
Experimental setup, design and feeding trial
 
The experiment was conducted for 60 days (July-September, 2023) in an ISW pond (10 ppt) of the Baniyani shrimp farm, ICAR-CIFE Rohtak Centre, Haryana. To conduct the feeding trial, 24 hapas (2.5 m x  2 m x 1 m) were installed in an optimally manured and fertilized ISW (10 ppt salinity) pond fortified with murate of potash. Juveniles of P. vannamei were stocked in hapas at the rate of 80 per hapa. The experiment followed a completely randomized design with eight experimental groups. The shrimps of all experimental groups were fed to satiation level four times (8.00, 12.00, 16.00 and 20.00 h) daily with respective diets. The hapas were cleaned every two days to remove the uneaten feed and algal debris to achieve uniform water circulation. Every fortnight, the shrimp weight was measured carefully to adjust the satiation level of feeding. The ISW quality parameters were analyzed daily according to APHA (2018). All the water quality parameters were within the optimum range during the entire experimental period of 60 days (Table 2).


 
Sampling
 
The shrimps’ initial and final live weight was measured using an electronic balance before commencement and after completion of the trial. The shrimps were kept unfed overnight before measuring the final body weight (for 22.00 h). Three shrimps from each replicate hapa were randomly collected, anaesthetized with ice-chilled water (Jana et al., 2021) and the hepatopancreas and muscle tissues were dissected in an ice-chilled condition. Tissues were then put into ice-cold phosphate buffer (0.025 M) solution and tissue homogenates (5%, w/v) were prepared using a mechanical homogenizer (REMI Equipments, India) for performing different enzyme assays. The entire process, starting from tissue collection, was carried out meticulously while maintaining a cold chain to preserve enzymatic activity. The tissue homogenates were centrifuged at 5000 rpm for 10 minutes at 4^oC using a refrigerated centrifuge. The resulting supernatant was transferred into 2 ml Eppendorf tubes and stored at -20^oC for further analysis.
 
Growth and nutrient utilization
 
The growth and nutrient utilization parameters were calculated using the following formulae:
 
Weight gain (g) = Final wet weight (g) - Initial wet weight (g)
 
The following formula calculates the metabolic growth rate of fish.
 

   

  
Where,
IWW = Initial wet weight (g).
FWW = Final wet weight (g).
ED = Experimental duration (days).
 

 

  

 

  
Enzyme assays
 
Tissue homogenate preparation
 
A 5% (w/v) tissue homogenate was prepared in an ice-cold phosphate buffer (0.025 M KH2PO4 , 0.025 M Na₂PO₄ ·12H‚ O, pH 7.5) using a mechanical tissue homogenizer (REMI Equipments, India). All steps were conducted under strict cold chain conditions from the tissue collection stage onward to maintain enzymatic activity. Using a cooling centrifuge, the homogenates were centrifuged at 5000 rpm for 10 minutes at 4^oC. The resulting supernatant was transferred into 2 ml Eppendorf tubes and stored at -20^oC.
 
Protein metabolic enzyme assays
 
The activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in muscle and hepatopancreas  tissues were measured using the method described by Wooten (1964). The substrates α-ketoglutarate and DL-aspartic acid were used for AST, while DL-alanine replaced DL-aspartic acid for the ALT assay. The optical density (OD) was measured at 540 nm relative to a blank.
 
Carbohydrate metabolic enzyme assays
 
The methodology of Wroblewski and Ladue (1955) was used to assess the lactate dehydrogenase (LDH) activity. Similarly, malate dehydrogenase (MDH) activity in hepatopancreas and muscle tissues was evaluated using the illustrated method of Ochoa (1955).
 
Statistical analysis
 
The data were analyzed using one-way ANOVA in SPSS software (version 22.0) to assess the overall effects of the means, following verification of normality and homogeneity of variance with Levene’s test. A 5% probability level (p<0.05) significant difference between the means was observed using Duncan’s multiple range test (DMRT) with post hoc analysis. The data analysis was displayed as mean ± standard error (SE).

Growth and nutrient utilization
 
The growth of animals, including fish, involves both muscle hypertrophy and hyperplasia, which are affected by the nutrients in their diet. Fish, including crustaceans, prefer protein over lipids and carbohydrates as the primary energy source. Protein being the costliest nutrient among others not only increases the feed cost but also results in the pollution of the culture environment via excretion of ammonia (Talukdar et al., 2021; Jana et al., 2022; Chuphal et al., 2024). Thus, using low-protein, high-energy diets can be a suitable dietary intervention strategy to improve production without compromising growth (Raghuvaran et al., 2023). Higher lipid utilization can be enhanced by using lipid utilizing nutraceuticals. Earlier studies evaluating individual effects of lipid-utilizing nutraceuticals such as taurine (Bhavatharaniya, 2021; Jana, 2022), L-carnitine (Raghuvaran et al., 2023) and Quercetin (Unpublished data) found that nutrient utilization can be improved without compromising growth. However, the information on the combinatorial effect of these nutraceuticals is still lacking. Hence, this study was conducted with this backdrop to assess the synergistic effect of these nutraceuticals. The growth and nutrient utilization parameters are given in Table 3. In the present study, the growth and nutrient utilization parameters, such as WG, MGR, TGC and FER, were significantly improved in all the nutraceutical-supplemented groups compared to the control group without nutraceuticals. Among the nutraceutical supplemented groups, treatment groups TC+T (50 % dose of L-carnitine and taurine) and TC+T+Q (33.3% of L-carnitine, taurine and quercetin) exhibited significantly (p<0.05) the highest WG, MGR, TGC and FER. Fish fed with nutraceuticals in combination could exhibit better growth due to their synergistic effects (Jin et al., 2019). Similarly, 0.5% dietary taurine (Bhavatharaniya, 2021) or 0.05% L-carnitine (Raghuvaran et al., 2023) or 0.04% quercetin (Unpublished data) improved the growth performance of P. vannamei reared in ISW, which had been reported in earlier studies. Additionally, dietary taurine (To et al., 2021; Shi et al., 2023; Mai et al., 2025), L-carnitine (Raghuvaran et al., 2023) and quercetin (Chen et al., 2023) mediated enhanced growth performance have been reported in P. vannamei. Our results corroborated the findings of Jana (2022), who also found that taurine and L-carnitine had a synergistic effect and performed better than their counterparts. Taurine facilitated lipid digestion and L-carnitine further transported the fatty acids in mitochondrial β-oxidation to satiate the energy demand of shrimp. Das (2024) reported similar findings, stating that feeding a combination of glycerol monolaurate and leonardite humic acid improved growth performance and lipid utilization.


 
Protein metabolic enzymes activity
 
Protein metabolic enzymes, such as AST and ALT, are crucial for transamination reactions, which facilitate the synthesis of new amino acids from existing ones (Knox and Greengard, 1965). In energy-sufficient conditions, these enzymes support the synthesis and accumulation of body proteins, contributing to growth. Conversely, they are involved in the oxidative degradation of proteins to generate energy during energy deprivation, as observed in various animals, including shrimp (Jiang et al., 2015; De Silva and Anderson, 1995). The supplementation of 0.05% L-carnitine and 0.5% taurine notably increased AST and ALT activities in P. vannamei juveniles reared in ISW (Raghuvaran, 2021; Bhavatharaniya, 2021). In our study, significantly (p<0.05), the highest AST and ALT activities in hepatopancreas and muscle were exhibited in treatment groups TC+T and TC+T+Q (Fig 1,2). The AST and ALT activities were non-significant (p>0.05) among treatment groups TC, TT, TQ, TC+Q and TT+Q. Individually, taurine and L-carnitine exhibited higher muscle AST and ALT activities than quercetin. However, all the nutraceutical groups showed significantly higher (p<0.05) activities than the control group without nutraceuticals.




 
Carbohydrate metabolic enzyme activity
 
LDH and MDH are enzymes involved in carbohydrate metabolism (Das, 2024; Paswan et al., 2025). Their activities increase in animals, including fish, under oxidative stress. During this condition, cytosolic LDH catalyzes the conversion of pyruvate into lactate through anaerobic glycolysis. The lactate is then converted into glucose in the liver via the Cori cycle and transported to muscles to sustain energy levels. Meanwhile, under oxidative stress-induced anaerobic conditions, mitochondrial MDH in the TCA cycle reduces oxaloacetate to malate. This malate moves to the cytoplasm, contributing to glucose synthesis through gluconeogenesis, ensuring a continuous energy supply. Due to their roles in stress response, LDH and MDH are considered biomarkers of oxidative stress (Raghuvaran et al., 2024; Mai et al., 2025). In the present study, significantly (p<0.05) higher LDH and MDH activities were expressed in the hepatopancreas and muscle of the control group (Fig 3, 4). All the experimental groups, except the control group, fed without nutraceuticals, exhibited significantly (p<0.05) lower LDH and MDH activities, indicating the stress-mitigating effect of the nutraceuticals. Both the TC+T and TC+T+Q groups exhibited significantly (p<0.05) lower MDH activity in hepatopancreas and muscle. However, in the case of LDH, similar activity was expressed among treatment groups TC+T, TC+T+Q, TC+Q and TT+Q.




 
Limitations
 
Although this study was conducted in hapas installed in ponds, a full-fledged feeding trial in ponds can be done in the future to validate the present findings.

In conclusion, all the nutraceuticals-fed groups exhibited improved growth and reduced stress compared to the control group. Among the nutraceutical-fed groups, both treatment groups, TC+T and TC+T+Q, showed higher growth, AST and ALT activities than other treatments. Similarly, lower LDH and MDH activities were found in the same treatment groups, indicating reduced stress compared to the control group. This study reveals that the taurine and L-carnitine combination is better than other nutraceutical combinations. The taurine and L-carnitine combination synergized, resulting in better nutrient utilization and reduced stress. This information will help formulate a low-cost, eco-friendly diet for P. vannamei reared in an ISW of 10 ppt salinity.

The authors declare that they have no conflict of interest to disclose.