Introduction

The worldwide consumption of edible fish grew at an annual rate of 3.0% from 1961 to 2019, nearly twice the rate of global population growth1,2. Aquaculture has developed quickly as a result of this increase in demand, especially through intensified culture, which reached a historic high of 87.5 million tonnes in 20202. Intense aquaculture accounted for 49.2% of worldwide seafood output.

Penaeus vannameiis a widely cultured crustacean all over the world due to its wide endurance to temperature and salinity3. Fish oil (FO) and fish meal (FM) are important dietary components and nutrient inputs used by feed manufacturers worldwide to prepare aquafeeds4. Although more sources with high protein are available, fishmeal is still recommended because of its high nutritional content, more easily absorbed crude protein and good balanced amino acid composition5. However, the use of fishmeal in diets is clearly trending lower due to factors such as rising global demand, rising costs, supply fluctuations, as well as sustainability difficulties6,7. Therefore, the quest for a substitute for fishmeal in shrimp diets has been the focus of numerous studies8,9. The amount of fishmeal produced over the last ten years has stabilized at about five million tons, and advancements in FM processing methods have made it easier to produce and distribute FO and FM that are obtained from fish byproducts2. The constrained global availability as well as the elevated price of fishmeal have led to a boom in the development and use of alternative protein sources over the past two decades10,11. Fishmeal has been replaced in shrimp diet by a wide range of different sources of protein that have been investigated until now. These include sources of protein found in plants, fisheries and aquaculture byproducts; land animal products such as insects and food industry coproducts; microalgae; low trophic species such as krill; and microbial biomass such as bacteria and yeast. In the production of shrimp feed, this might decrease our dependence on FM12.

As the demand and cost of conventional feed ingredients like fishmeal continue to rise, there is an increasing need to search for alternative sources with nutritional values comparable to fishmeal. Novel feed ingredients refer to alternative ingredients used in animal feed that are obtained from non-conventional sources. These ingredients are incorporated into animal diets to supplement conventional feed ingredients, aiming to improve sustainability, reduce costs, and meet the nutritional requirements of shrimp. Research on novel feed ingredients is focused on enhancing the efficiency and environmental sustainability of animal production. However, limited research has been conducted in this area13,14,15. By identifying viable alternatives, we can reduce much dependence on conventional ingredients like fishmeal16. While unconventional feed research has a long history, current efforts are intensifying worldwide, driven by both researchers and industries. They are developing novel ingredients based on circular economy principles to maximize resource efficiency and achieve zero waste in the agro-food value chain while emphasizing cost-effectiveness. Instead of replacing fishmeal with a single ingredient, the emerging approach combines various novel ingredients to meet animal’s dietary needs. The nutritional content of shrimp may be better when different novel feed ingredients are combined than when they are used individually.

The novel feed ingredients chosen for this research include poultry byproduct meal (PBM), insect meal (IM), fish waste (FW) (by-products such as discarded fish fillets, trimmings, and offal), rapeseed meal (RM), peanut meal (PM), and single-cell protein (SCP). These ingredients were combined using a concept that integrates plant, animal, and microbial-based ingredients12,17,18 at different inclusion levels. The various inclusion levels of the novel ingredient combination in the experimental diets are as follows: Diet 1 contains 248.2 g, Diet 2 contains 288.2 g, Diet 3 contains 328.2 g, and Diet 4 contains 368.2 g of the novel ingredient combination per kg of experimental diet.

Insect meal is a sustainable and cost-effective alternative to fish meal and plant-based diets in shrimp farming. It requires minimal land, water, and energy and serves as a highly viable protein source, rich in essential amino acids, under controlled farming conditions19,20,21. Poultry by-product meal (PBM) is rich in protein and essential amino acids, except for lysine and methionine22,23,24. Peanut meal, known for its high protein and arginine content, offers a palatable and economical substitute for fish meal and soybean meal in aquafeeds25,26. Rapeseed meal is a promising alternative to other plant proteins in aquafeeds due to its balanced amino acid profile, global availability, and cost-effectiveness27. Single-cell proteins (SCPs), derived from microorganisms such as microalgae, yeast, and bacteria, help reduce environmental waste and offer an effective alternative to conventional protein sources in aquaculture28,29. Fish waste (discarded fish fillets, trimmings and offal) biomass is a rich source of bio functional compounds such as omega-3 oils, enzymes, polysaccharides, gelatin, and bioactive peptides30,31, although its biochemical composition, including protein, ash, and lipid levels, varies depending on the source species32.

Additional protein sources were used in place of FM in practical diets by combining novel feed ingredients at different inclusion levels that were developed specifically for this study. This helped to compensate for nutrient deficiencies in a single ingredient and also leads the better nutritional value of novel protein sources. It is important to maintain nutritional balance in FM-free diets, as demonstrated by this study on analysing the utilization of novel protein sources at different inclusion levels in shrimp feed. It highlights areas for further research on FM replacement and points out the limitations of the current studies. Therefore, this research was done to assess the effects of diets formulated with different inclusion levels of novel ingredient combination on growth performance, whole body proximate composition, apparent digestibility, digestive enzymes and gene expression of P. vannamei.

Materials and methods

Shrimp and experimental conditions

A 32 m³ nursery tank with continuous aeration was used to raise 1,000 post-larvae (PL 12) of Penaeus vannameito the juvenile stage (1 g). The larvae were obtained from Star Aqua Hatchery in Koovathur, Chengalpattu, Tamil Nadu, India. The shrimp were fed a commercial diet (Royal Dragon DT311, Sheng Long Biotech International Co., Ltd., India) containing 37% protein at four intervals a day (at 9:00, 10:00, 14:00, and 18:00 h) throughout their acclimatization. An eight-week feeding trial was carried out at the wet lab of the Institute of Fisheries Post Graduate Studies, TNJFU, Vaniyanchavadi, India. After proper acclimatization, the shrimp were gradually introduced into the experimental tanks, where shrimp in the treatment groups were fed the prepared diets till apparent satiation four times daily (09:00, 10:00, 14:00, and 18:00 h). Weekly checks were conducted on shrimp weight, survival, and health. Feed rations were adjusted based on weight to minimize uneaten feed. Brackish water from the wet lab (salinity: 15 ± 1 ppt) was used, with water exchanges performed every third day. Daily monitoring of water quality during the trial recorded the following values: salinity (15.01 ± 0.71 ppt) was measured with a refractometer, temperature (28.43 ± 0.50 °C) measured with a digital thermometer, dissolved oxygen (6.21 ± 0.81 ppm) measured with a dissolved oxygen (DO) meter, and pH (8.24 ± 0.43) measured with a pH meter. Total ammonia N (0.01 ± 0.01 ppm), nitrite-N (0.05 ± 0.01 ppm), and nitrate N (10 ± 0.1 ppm) were measured according to APHA33 standards.

Diet preparation

The formulation and the nutrient composition of the experimental diets are shown in Table 1. Four isonitrogenous (crude protein, 36%), isolipidic (crude fat, 6%), and isoenergetic (gross energy, 16 MJ/Kg) diets were formulated with ingredients, namely poultry byproduct meal (PBM), insect meal (IM), rapeseed meal (RM), peanut meal (PM), fish waste (FW), and single-cell protein (SCP), at 24.82% (Diet 1), 28.82% (Diet 2), 32.82% (Diet 3), and 36.82% (Diet 4). The dry feed components were all finely pulverized in a pulverizer, passed through a 180-micron mesh screen, and mixed well. Then, the ground ingredients were combined with the oil sources (fish oil, soy lecithin), all of the additives, and the necessary amount of water. The mixture was vigorously mixed for 15 min using an electric blender (Gaocheng-GC-MT1200, Mogli Labs, India Pvt. Ltd.) to achieve homogeneity. The feed included a chromic oxide marker so that the apparent digestibility coefficient (ADC) could be determined. The soft dough was properly mixed and then cooked for 15 min at 80 °C. After cooling, the soft dough was pelletized using a 1.6 mm die in a tabletop pelletizer. The pellets were then air-dried for 12 h at 45 °C to achieve the appropriate moisture content. At last, every dried feed was put into an airtight plastic container and kept at 4 °C until needed.

Table 1 Ingredient and nutrient composition (% dry weight basis) of diets formulated with different inclusion levels of novel feed ingredients.
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Growth parameters and sample collection

Shrimp were mass-weighed, counted, and anesthetized with MS-222 (Sigma-Aldrich Inc.) after the growth trial was over. Calculations were made for mean final body weight, weight gain, FCR, SGR, ADG, PER, and survival rate. For the purpose of gene expression analysis and digestive enzyme assays, nine shrimp per treatment (three per replicate) were chosen at random, and the remaining shrimp were utilized for evaluation of whole-body chemical composition. The growth parameters computed on the basis of the collected data were listed below:

$$\begin{aligned} {\text{Weight}}\;{\text{gain}}\;\left( {{\text{WG}}} \right)\left( {\text{g}} \right) & = {\text{Final}}\;{\text{weight}}\left( {\text{g}} \right){-}{\text{Initial}}\;{\text{weight}}\left( {\text{g}} \right) \\ {\text{Survival}}\;{\text{rate}}\left( {{\text{SR}}} \right)\left( \% \right) & = ({\text{Total}}\;{\text{shrimp}}\;{\text{at}}\;{\text{final}} \\ & \quad /{\text{Total}}\;{\text{shrimp}}\;{\text{at}}\;{\text{initial}}) \times {\text{1}}00 \\ {\text{Average}}\;{\text{daily}}\;{\text{growth}}\left( {{\text{ADG}}} \right)\left( {\text{g}} \right) & = \left( {{\text{Final}}\;{\text{weight}}\left( {\text{g}} \right) - {\text{Initial}}\;{\text{weight}}\left( {\text{g}} \right)} \right) \\ & \quad /{\text{Total}}\;{\text{number}}\;{\text{of}}\;{\text{days}} \\ {\text{Feed}}\;{\text{conversion}}\;{\text{ratio}}\left( {{\text{FCR}}} \right) & = {\text{Total}}\;{\text{feed}}\;{\text{consumed}}\left( {\text{g}} \right) \\ & \quad /{\text{Total}}\;{\text{weight}}\;{\text{gain}}\;{\text{of}}\;{\text{shrimp}}\left( {\text{g}} \right) \\ {\text{Specific}}\;{\text{growth}}\;{\text{rate}}\left( {{\text{SGR}}} \right)\left( {\% /{\text{day}}} \right) & = \left[ {\left( {{\text{log}}_{{\text{n}}} \;{\text{final}}\;{\text{weight}}} \right.} \right. \\ & \quad \left. { - {\text{log}}_{{\text{n}}} {\text{initial}}\;{\text{weight}}} \right)\left. {/{\text{total}}\;{\text{days}}} \right] \times {\text{1}}00 \\ {\text{Protein}}\;{\text{efficiency}}\;{\text{ratio}}\left( {{\text{PER}}} \right) & = {\text{Total}}\;{\text{weight}}\;{\text{gain}}\;{\text{of}}\;{\text{shrimp}}\left( {\text{g}} \right) \\ & \quad /{\text{Total}}\;{\text{protein}}\;{\text{consumed}}\left( {\text{g}} \right). \\ \end{aligned}$$

Proximate analysis

Standard methods were used to evaluate the dry matter, crude protein, ether extract, and ash in the diets, whole body, and faeces, according to AOAC34. A final whole-body chemical composition assessment was conducted on 10 randomly selected shrimp from each tank (30 shrimp per treatment) at the termination of the growth trial.

Faeces collection

The control and experimental diets were hand-fed to the shrimp four times daily at 09:00, 10:00, 14:00, and 18:00 h, until the shrimp appeared satiated. After a one-week acclimation period, faecal samples were collected according to the method described by Lin et al.35. After each feeding, one hour later, unconsumed leftover feed and faeces were disposed of. Faecal samples were drawn twice a day at 12:00 and 16:00 h from every replicate. The samples were then combined according to treatment, oven-dried, and kept for chemical examination.

Determination of digestibility

Chromic oxide (Cr2O3), an inert marker, was used to calculate the ADC, or apparent digestibility coefficient, of the diets in the study at a dose of 5 g/kg of feed. An inductively coupled plasma atomic emission spectrometer was used to determine the amount of chromium present in diets and faecal samples. In accordance with method of Cho et al.36, the apparent digestibility coefficient (ADC) of diets’ dry matter, protein, and lipid was computed as follows:

$$\begin{aligned} {\text{ADC}}\;{\text{of}}\;{\text{dry}}\;{\text{matter}}\left( \% \right) & = {\text{1}}00 - [{\text{1}}00 \times (\% {\text{Cr}}_{{\text{2}}} {\text{O}}_{{\text{3}}} \;{\text{in}}\;{\text{feed}} \\ & \quad / \% {\text{Cr}}_{{\text{2}}} {\text{O}}_{{\text{3}}} \;{\text{in}}\;{\text{faeces}})] \\ {\text{ADC}}\;{\text{of}}\;{\text{protein}}\left( \% \right) & = {\text{1}}00 - [{\text{1}}00 \times (\% {\text{Cr}}_{{\text{2}}} {\text{O}}_{{\text{3}}} \;{\text{in}}\;{\text{feed}}/ \% {\text{Cr}}_{{\text{2}}} {\text{O}}_{{\text{3}}} \;{\text{in}}\;{\text{faeces}}) \\ & \quad \times (\% {\text{protein}}\;{\text{in}}\;{\text{faeces}} / \% \;{\text{protein}}\;{\text{in}}\;{\text{feed}})] \\ {\text{ADC}}\;{\text{of}}\;{\text{lipid}}\left( \% \right) & = {\text{1}}00 - [{\text{1}}00 \times (\% {\text{Cr2O3}}\;{\text{in}}\;{\text{feed}} / \% {\text{Cr2O3}}\;{\text{in}}\;{\text{faeces}}) \\ & \quad \times (\% {\text{lipid}}\;{\text{infaeces}} / \% \;{\text{lipid}}\;{\text{in}}\;{\text{feed}})] \\ \end{aligned}$$

Quantification of Digestive enzymes

After removing the hepatopancreas from three shrimp and midgut of intestine tissue samples from five shrimp, the materials were homogenized with a 0.25 M sucrose solution and centrifuged for 10 min at 6000 rpm. For the examination of digestive enzymes, such as lipase, amylase, and protease, the supernatant was maintained at −20°C. Lipase activity was measured as the amount of NaOH needed to neutralize released fatty acids per minute of incubation, following the method of Cherry and Crandall37. Protease activity was measured as µmol of tyrosine released per milligram of protein per minute, with casein serving as the substrate, according to Drapeau38. Amylase activity was measured using 1% starch and DNS according to Rick and Stegbauer39, quantified by a maltose standard curve, and expressed as µmol of maltose per minute per mg of protein. Total protein content was determined following the method of Bradford40.

Quantitative real time PCR (qRT-PCR)

Hepatopancreas tissue from three shrimp in each group was collected at the end of the experiment under cold, sterile conditions and preserved at −80 °C to analyse the gene expression of IGF-I and IGF-II. As directed by the manufacturer, total RNA was extracted using TRIzol (easy-RED). Samples were used for cDNA synthesis after RNA quality (A260/A280 ratio ≥ 1.8) was assessed using NanoDrop. DNase-treated RNA, reverse transcriptase, and a heat cycler were used in the PCR amplification process. Melting curve analysis (62–95 °C) was used for real-time PCR. Using the 2-ΔΔCt technique according to Livak and Schmittgen41, gene expression was measured and normalized to β-actin. Table 2 lists the primers that were used in the gene expression.

Table 2 Primer sequences for real-time PCR used for gene-expression analysis.
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Table 3 Growth performance and nutrient utilization of P. Vannamei fed diets formulated with different inclusion levels of novel feed ingredients.
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Table 4 Whole-body proximate composition (% wet weight basis) of P. Vannamei fed diets formulated with different inclusion levels of novel feed ingredients.
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Table 5 Apparent digestibility coefficients of dry matter, crude protein and crude lipid of P. Vannamei-fed diets formulated with different inclusion levels of novel feed ingredients.
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Table 6 Digestive enzyme activities (U mg−1 protein) in hepatopancreas of P. Vannamei-fed diets formulated with different inclusion levels of novel feed ingredients.
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Table 7 Intestinal digestive enzyme activities (U mg−1 protein) of P. Vannamei-fed diets formulated with different inclusion levels of novel feed ingredients note: the data are represented as mean ± SD of three replicates per treatments (n = 3) and the values with different superscripts indicate significant differences as determined by Duncan’s test (p < 0.05).
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Fig. 1
figure 1

Growth-related gene expression (IGF-I) in the hepatopancreas of P. vannamei fed diets formulated with different inclusion levels of novel feed ingredients. Bars with different letters are significantly different (p < 0.05).

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Fig. 2
figure 2

Growth-related gene expression (IGF-II) in the hepatopancreas of P. vannamei fed diets formulated with different inclusion levels of novel feed ingredients. Bars with different letters are significantly different (p < 0.05).

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Statistical computation

To find significant differences between treatments, all findings were submitted to a one-way analysis of variance, which was followed by Duncan’s multiple range tests using SPSS version 20 (SPSS Inc., Chicago, IL, USA). When a P value was 0.05 or below, the treatment effects were deemed significant.

Ethical approval

The Institutional Animal Ethics Committee (IAEC) of Tamil Nadu Dr. J. Jayalalithaa Fisheries University in Nagapattinam has approved this research under the grant number 3/1128/IAEC/TNJFU/IFPGS.

Results

Growth performance and feed utilization

The growth performance of P. vannamei is summarized in Table 3. Shrimp fed diet 3 and 4 showed significantly higher final weight, weight gain, average daily growth, and specific growth rate (p < 0.05). However, no significant differences were observed in survival, PER, or FCR (p > 0.05) of P. vannamei fed diets formulated with different inclusion levels of novel ingredient combination.

Whole-body proximate composition

The whole-body proximate composition of P. vannamei is presented in Table 4. No statistically significant difference was found in the whole-body proximate composition, such as crude protein, crude lipid, moisture, and ash (p > 0.05) of P. vannamei fed diets formulated with different inclusion levels of novel ingredient combination.

Determination of digestibility

Apparent digestibility coefficient (ADC) of dry matter, crude protein, and crude lipid of Penaeus vannamei are shown in Table 5. The results indicate that there was no statistically significant difference in the digestibility of dry matter, crude protein, or crude lipid (p > 0.05) among shrimps fed diets formulated with different inclusion levels of novel ingredient combination.

Digestive enzyme activity

The activities of digestive enzymes, including amylase, protease, and lipase, in the hepatopancreas of Penaeus vannamei are shown in Table 6. The amylase and protease activities showed no statistically significant difference (p > 0.05) among shrimp fed diets formulated with varying inclusion levels of novel ingredient combination. In contrast, lipase activity was significantly higher in shrimp fed diet 4, which was not significantly different from diet 3 (p < 0.05).

The digestive enzyme activities, including amylase, protease, and lipase, in the intestine of Penaeus vannamei are shown in Table 7. The amylase activity was significantly increased in shrimp fed diet 3 and 4 than in shrimp fed with diet 1 (p < 0.05). Shrimp-fed diets 3 and 4 have shown significantly higher lipase activity compared to other diets, whereas protease activity was found with no significant difference in shrimp-fed diets formulated with varying inclusion levels of novel ingredient combination.

Growth gene expression

Growth genes such as IGF-I and IGF-II expression in Penaeus vannamei are illustrated in Figs. 1 and 2. IGF-I and IGF-II were neither upregulated nor downregulated (i.e.,) found with no significant difference (p < 0.05), in shrimp-fed diets formulated with different inclusion levels of novel ingredient combination.

Discussion

Several studies have examined the effects of incorporating individual ingredients with a nutritional profile nearly equivalent to fishmeal42,43, but there is limited research on the use of a combination of animal, plant, and microbial-based ingredients14,15. The purpose of this experiment was to investigate the impact of different inclusion levels of this novel ingredient combination. Supporting the results of better growth performance and nutrient utilization, shrimp fed Diet 4 exhibited significantly higher RBA and proPO activity (p> 0.05), indicating enhanced growth without compromising their health and immune status. The improved growth may also be attributed to the presence of balanced amino acids and fatty acids in Diet 4. Incorporating a single ingredient into a diet may sometimes lead to negative effects, so the concept of using a combination of ingredients helps mitigate these issues13,14. Studies have demonstrated that the use of ingredient combination, even at high inclusion levels44,15, can improve the growth and health of shrimp. In line with the results of previous studies and the findings of this study, the growth parameters observed suggest that Diet 4, formulated with a combination of novel feed ingredients (PBM, IM, FW, RM, PM, and SCP) at an inclusion level of 36.82%, effectively meets the nutritional requirements of shrimp without compromising their growth or nutrient utilization.

Whole-body carcass composition mostly depends on the nutrient composition of diets. The components of proximate, such as moisture, fat, protein, and ash, are crucial markers of an organism’s metabolic state. Higher protein and lipid content generally reflects greater energy density45. However, these components can differ widely depending on species, size, sex, feeding season, and physical activity46. In the present research, the whole-body proximate composition of P. vannameialigns with the findings of Manikandan and Felix18, who supplemented plant-based diets containing a corn gluten meal and soybean meal protein blend with dietary L-lysine and phytase to enhance the growth performance of P. vannamei and reported no significant differences in the shrimp’s proximate composition.

The breakdown and absorption of food and its nutrients depend critically on the activity of digestive enzymes47,48. According to earlier research49,50,51, diets formulated with varying inclusion levels of novel ingredient combination can either positively or negatively alter the activities of digestive enzymes. The present research revealed no significant alterations in the amylase and protease enzyme activities of the hepatopancreas (p > 0.05). In the same way, no statistically significant difference was noted in the intestinal protease activity (p > 0.05) of P. vannamei. On the other hand, the inclusion level of novel ingredient combination in diet 3 (32.82%) and diet 4 (36.82%) of P. vannamei was significantly higher in terms of hepatopancreas lipase activity (p < 0.05) and the same was found in intestinal amylase and lipase activities. These results can be correlated with the different digestibility, such as crude protein, crude lipid and dry matter digestibility, which has shown no significant difference (p > 0.05) in shrimp-fed diets formulated with different inclusion levels of novel ingredient combination in P. vannamei. The results of the digestive enzyme analysis in the present research showed a clear correlation with digestibility, indicating that the enzyme activity aligns closely with the observed digestibility findings. Therefore, the combination of ingredients, even at an inclusion level of 36.82%, did not adversely affect the nutrient digestion and absorption in P. vannamei, and all these were reflected in the results obtained in the present study.

Digestibility of feedstuffs is essential for assessing their utilization52. The amount of feed sample that is absorbed in an animal’s digestive tract is indicated by its digestibility35. In high-density culture conditions, where leftover that are not properly assimilated can lead to water pollution, higher water treatment costs, and increased susceptibility to shrimp illnesses and mortalities. Therefore, extremely digestible feedstuffs are particularly significant53. Determination of apparent digestibility of ingredients is crucial for evaluating the potential of novel protein sources54. Enhancing apparent digestibility coefficients (ADCs) through research and innovation is vital for the sustainable growth of aquaculture feed. In this study, no significant differences were found in the digestibility of crude protein, crude lipid, and dry matter (p> 0.05). This can be linked to the activity of digestive enzymes, such as amylase and protease in the hepatopancreas and protease in the intestine. These findings suggest that the digestive efficiency of shrimp fed diets with varying inclusion levels of novel ingredient combination was positively influenced by their nutritional composition, including the presence of balanced amino acids and fatty acids, which have beneficial effects55,56,57. Therefore, the observed trends in digestibility and the nutritional profile of the diets in the present study support the conclusion that the combination of novel feed ingredients, even at an inclusion level of 36.82%, did not negatively impact the digestibility capacity of P. vannamei.

Over the last decade, the inclusion levels of various feed ingredients have been researched according to growth statistics of animals used for research. The GH/IGF-Iaxis is a key signalling pathway for growth and nutrient distribution58 and plays roles in muscle differentiation, metabolism, behaviour, and immunity. In aquatic animals, dietary modifications can be modulated by the GH/IGF-I axis due to IGF-Imetabolic functions59. In this research, the IGF-I and IGF-II gene expression activities were not significantly (p > 0.05) affected by the diets formulated with different inclusion levels of novel ingredient combination in P. vannamei. These results could be attributed to better nutrient utilization, and required amino acids and fatty acids were met by the diet fed to shrimp. This result concluded that even at a 36.82% inclusion level of novel ingredient combination, the growth of P. vannamei was not negatively influenced.

Conclusion

The outcomes of the present research indicated that different inclusion levels of novel ingredient combination did not compromise the growth performance, whole-body composition, digestibility, digestive enzymes, and gene expression activities of P. vannamei. The 36.82% inclusion level of novel ingredient combination has improved the growth performance and nutrient utilization of P. vannamei without compromising growth and nutrient utilization. These findings suggest that alternative dietary formulations can effectively replace conventional ingredients, and furthermore, this study provides insights into the feasibility and sustainability of incorporating novel feed ingredient combination at varying inclusion levels in P. vannamei diets to optimize growth, nutrient utilization, and immune response, paving the way for improved shrimp feed formulations.