Growth, survival, nutritional value and phytochemical, and antioxidant state of Litopenaeus vannamei shrimp fed with premix extract of brown Sargassum ilicifolium, Nizimuddinia zanardini, Cystoseira indica, and Padina australis macroalgae

The effect of including the premix extract of the brown Sargassum ilicifolium, Nizimuddinia zanardini, Cystoseira indica, and Padina australis (MPE) macroalgae in the diet on the growth performance, survival, nutritional values, phytochemical, and antioxidant state of whiteleg shrimp (Litopenaeus vannamei) was investigated in this study, where in 1200 post larvae with an average weight of 57.53 ±\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm$$\end{document} 0.47 mg were distributed randomly by categorizing 100 pieces into 4 groups (three tank iterations per group). The control group was only fed with the concentrated feed (MPE0), while the other groups were fed with a basic diet that consisted of MPE5 (MPE5), 10 (MPE10), and 15 (MPE15) g kg−1 of feed for 8 weeks. According to the results, the highest final weight, specific growth ratio, weight gain, and protein efficiency ratio were recorded for the MPE15 group. The lowest feed conversion ratio was observed in the group fed with MPE15 (P < 0.05). The highest level of crude protein was recorded for the groups fed with MPE10 and MPE15 (P < 0.05). The highest amount of PUFA, total sterols, phenol, flavonoid, catalase (CAT), and glutathione was observed in shrimps fed with MPE15. The results are generally indicative of the positive effect of the premix aqueous extract of the brown S. ilicifolium, N. zanardini, C. indica, and P.australis macroalgae at the 15 g kg−1 of MPE feed level on the growth, nutritional value and phytochemical, and antioxidant status of L. vannamei.


Introduction
Shrimp breeding has recently gained considerable importance in developing countries, and effective measures have been taken to improve shrimp breeding and production due to the problems caused by overharvest of natural resources (Alam et al. 2007; Mousavi Nadushan and Hellat 2019). In our country, Iran, 1800 km of the southern coasts and a fraction of 900 km of the northern coasts have the potential for the creation of shrimp farms. The low survival rate of larvae is among the problems challenging shrimp breeding centers. Therefore, proper nutrition management can contribute to the achievement of improvements in larval breeding conditions .
In recent years, many studies have been conducted on the use of a wide range of algae species as sources of protein in the diet of aquatic animals. Nutritional studies centered on diets consisting of seaweed or seaweed extracts have mostly revealed that the level of these nutrients in the feed is so low (less than 10%) that they fail to demonstrate their potential benefits as functional (binding effect), nutritional, and food-pharmaceutical (health protective effect) supplements in shrimps feed (Cruz-Suárez et al. 2008). The adequate amount of these materials in shrimp feed varies by the algae or the feeding species. The use of algae in the feed often improves feed quality (water stability, water holding capacity, and texture), increases feed intake and feed productivity, and improves the growth performance and the quality of animal products, more pigmentation, and lower cholesterol contents (Akbary and Shahraki 2020;Akbary et al. 2021a). Some macroalgae (Ulva Undaria, Ascophyllum, Porphyra, Sargassum, Polycavernosa, Gracilaria, and Laminaria) are commonly included in shrimp feed (Gutiérrez-Leyva 2006;Cruz-Suárez et al. 2009;Akbary and Aminikhoei 2018a).
Algae can be classified into two groups, namely the macroalgae and the microalgae. Marine macroalgae are photosynthesizing plants that constitute the primary biomass in intertidal regions. There are approximately 9000 macroalgae species, which are generally classified into three main groups, namely brown algae (Phaeophyta), red algae (Rodophyta), and green algae (Chlorophyta) based on their pigments. Recently, algae have been increasingly used in aquaculture because they contain nutrients such as antioxidants, essential fatty acids (ω 3 and ω6), essential amino acids, vitamins, minerals, carbohydrates, and beta-carotene (Rajapakse and Kim 2011;Arumugama et al. 2017). The inclusion of algae in the diet of aquatic animals not only reduces the feeding costs but also improves the efficiency of aquatic nutrition and digestion, thereby reinforcing the fish immune system (Tabarsa et al. 2012). Besides, it affects water quality by improving the efficiency of digestion (Mitra et al. 2010). The levels of protein and essential amino acids in macroalgae vary largely, and polysaccharides and phenolic compounds in certain species may affect protein digestibility. Therefore, it cannot be concluded that all macroalgae can serve as sources of protein. Many species also contain insignificant amounts of digestible proteins that can replace the sources of protein in animal feed (Øverland et al. 2019).
Phenolic compounds are secondary production metabolites, which are synthesized through shikimic acid and phenylpropanoid pathways (Santos-Sánchez et al. 2019). Phenolic molecules are widely released by algae to mainly serve protective, structural, and ecological purposes. The amount of phenolics in brown macroalgae exceeds that of red and green macroalgae (less than 10-140 g kg −1 of dry matter) (Shi et al. 2005). These compounds are typically used to prevent lipid per oxidation and increase shelf life (Holdt and Kraan 2011).
The antioxidant defense enzymes, which are used as biomarkers for shrimps, are involved in the removal of free radicals (Akbary and Aminikhoei 2018c;Akbary et al. 2021a).
The positive effect of various macroalgae on the growth, body chemical composition, and fatty acids and amino acids of different shrimp and fish species has been explored in numerous studies (Ju et al. 2009;Choi et al. 2014;Anaya-Rosas et al. 2019;). The antioxidant state in the gray mullet (Mugil cephalus) (Akbary and Aminikhoei 2018b) and the European bass, Dicentrarchus labraxm (Peixoto et al. 2016), sterols in brown macroalgae such as Stoechospermum marginatum, P.australis (Akbary et al. 2021b), and Pelvetia siliquosa (Rotini et al. 2013), and phenol content and antioxidant activity in macroalgae such as Padina australis, Stoechospermum marginatum, and Ahnfeltiopsis pygmaea macroalgae on Chahbahar coasts have also been investigated (Akbary et al. 2021c), Hizikia fusiformis (Kolanjinathan et al. 2009). For instance, Akbary and Aminikhoei (2018b) reported that the inclusion of different levels (5, 10, and 15 mg kg −1 food) of Ulva rigida extract in the grey mullet diet significantly increased superoxide dismutase activity and reduced glutathione. Peixoto et al. (2016) also indicated that the use of a food supplement containing Ulva spp., Gracilaria spp., and Fucus spp. increased the level of glutathione reductase (GR) in the European bass, D.labrax.
Researchers and breeders are searching for novel and better solutions for the realization of the aquaculture goal given the high distribution of macroscopic algae on the coasts of the Persian Gulf and the Sea of Oman, and the fact that sustainable and successful aquaculture is contingent on protecting the health of aquatic species and creating the ideal breeding conditions for maximizing the growth of the aquatic species and lowering the secondary costs of production.
The present study was an attempt to investigate the effect of the aqueous premix extract of the brown Sargassum ilicifolium, Nizimuddinia zanardini, Cystoseira indica, and Padina australis macroalgae on the growth performance, survival, nutritional value, phytochemical, and antioxidant state of L. vannamei.

Preparation of the macroalgae premix extract and experimental diets
Seaweed biomass samples of three species, S. ilicifolium, N. zanardini, C. indica, and P. australis were collected from the coasts of Chabahar coasts in December 2021 during the tides and following their transfer to the laboratory, they were washed several times with fresh water until the epiphyte organisms and mud were completely removed from them. Thereafter, they were dried in the shade at the room temperature (25 °C). After the macroalgae was ground with an electric grinder, a mixed powder of four types of macroalgae (1:1:1:1) was prepared and stored at − 4 °C until the next time of use (Choi et al. 2015). To prepare an aqueous extract from the macroalgae premix (three iterations), 5 g of the powder obtained from the premix was mixed with 100 mL of water and was properly shaken for 20 min. Next, the containers were closed and kept in the dark for 72 h, and then the solution on the top was collected carefully and the extract was concentrated for 6 h in a rotary evaporator (IKA, Germany) at a temperature of 45 °C after it passed through a filter paper (Whatman paper no. 1). Finally, the resulting extract was placed in a clean Petri dish under a laminar hood until the rest of the solvent evaporated. It was stored at − 20 °C until the next time of use (Choudhury et al. 2005). The premix extract of macroalgae (MPE) was added as an edible additive to the experimental diets.
After MPE was mixed with the nutrients, oil and 30% distilled water were added. The resulting paste was pelleted into 1 mm particles and dried using a chopper and then it was stored at a temperature of − 20 °C (Choi et al. 2015). The 0, 5, 10, and 15 g kg −1 of diet of the MP extract (MPE 0 , MPE 5 , MPE 10 , and MPE 15 ) were added to the basic control diet (Table 1). Table 2 presents the assessment of the nutritional value, phytochemical, and antioxidant activity of MPE. The studied nutritional value of the MP extract includes the chemical composition (AOAC 2000), amino acids (Lindroth and Mopper 1979), and fatty acids (Pal et al. 2013). The resulting premix extract was examined for its phytochemical compounds using various reagent tests. The studied phytochemical compounds including sterols were measured through gas chromatography (Unicam 4600, Germany) (Liu et al. 2007) and phenol and flavonoid (Ebrahimzadeh et al. 2008) were measured by a spectrophotometer (Shimadzu, uv-1800, Japan), which were read at the 765 and 415 nm wavelengths, respectively. The assessment of antioxidant activity was conducted using the diphenylpicrylhydrazyl premix extract of macroalgae with a free radical neutralization capacity measurement kit and the DPPH technique (Ziotex kit from Kavosh Azma Company) at a wavelength of 515 nm with the aid of a micromole spectrophotometer (Shimada et al. 1992).

Shrimp and experiment design
A total 1200 Pacific white shrimp larvae with an average weight of 10 ± 0.00 mg were purchased from Ajdari Shrimp Breeding Center (Kenarak, Iran) and were dispatched to Chahbahar Far Water Fisheries Research Center in Iranian double-walled bags (filled with two-thirds air and one-third water). To enable the shrimps to adapt to the laboratory conditions, they were stored for 2 weeks in two 300-L tanks with continuous aeration and were Artemia urmiana nauplii with a density of 1 to 19 mL every 4 h (four times a day) until they were full. Following 2 weeks of adaptation, the post larvae with an average weight of 57.53 ± 0.47 mg were randomly distributed in 12 plastic tanks (60 L) with a density of 100 pieces (four groups with three replication). This stage also involved continuous aeration and daily replacement of 30% of the water in each tank. The control group was fed only the concentrated food (MPE 0 ) while the other groups were fed on a basic diet containing MPE 5 (MPE 5 ), 10 (MPE 10 ), and 15 (MPE 15 ) g kg −1 of feed four times a day (8:00, 12:00, 16:00, and 20:00) for 60 days. Biometry (measurement of weight and length) of shrimps was carried out every 15 days to calculate the amount of feed for the total biomass weight in each tank (Akbary et al. 2020b). In the course of the experiment, the physical and chemical conditions of water including temperature were measured by a mercury thermometer with an accuracy of 0.1 °C. Besides, dissolved oxygen was measured using a digital oxygen meter (TECPEL DO-1609), and PH was measured electrically on a daily basis (Ebro, PHT-3140). The average water temperature, oxygen level, acidity, and salinity were maintained at 30 ± 2 °C, 8.2 ± 0.5 mg L −1 , 7.5 and 35 ± 0.47 g L −1 , respectively. 2.20 ± 0.34 Carbohydrate (g 100 g −1 ) 5.10 ± 0.89 Raw energy (cal g −1 ) 4238.86 ± 358 Moisture (g 100 g −1 ) 85.46 ± 4.28 TAA a (g amino acid 100 g −1 ) 19.29 ± 5.23 SFA b (% of total fatty acid) 40.90 ± 15.13 USFA c (% of total fatty acid) 11.70 ± 0.83 PUFA d (% of total fatty acid) 47.4 ± 8.12 Sterol (g 100 g −1 ) 234.54 ± 12.18 Phenol (mg of GAE g −1 of extract) 83.46 ± 7.07 Flavonoid (mg QE g −1 extract) 10.01 ± 0.98 Diphenylpicrylhydrazyl(μmolTrolex g −1 of extract) 1323.78 ± 11.28

Measuring shrimp growth performance
The length and weight of all shrimps were measured with an accuracy of 1 mm and 0.001 g at the end of the experiment period (60 days), respectively. The final weight (FW), percentage of weight gain (WG), survival, feed conversion ratio (FCR), specific growth ratio (SGR), and protein efficiency ratio (PER) are reflected in relations 1 to 5 based on data resulting from the biometry (Harikrishnan et al. 2011).
WI initial weight) g),Wf: final weight (g), t: Length of breeding period (day) Wf final weight (g), Wi: initial weight(g) F the amount of food consumed (g), WG: acquired weight BWf final weight (g), BWi: initial weight (g), AP: protein intake per fish)

Chemical composition
The chemical composition and raw energy analysis of the tested shrimp were conducted according to the standard method of AOAC (2000) Briefly, 3 pieces of shrimp were randomly selected from each replicate and oven dried for 24 h at 60 °C (AOAC 2000). Then the dry mass was removed from the oven, ground into fine powders with a mortar and pestle, and kept in closed containers in the freezer until further use. Total carcass protein was measured by employing the Kjeldahl procedure, total fat by Soxhlet extraction procedure and ether solvent, and finally moisture by placing the sample at 105 °C, weighing the mass after drying, and then the ash obtained from burning the sample at 550 °C for 6 h.

Fatty acid profile
Initially, 100-200 mg of dried shrimp powder was placed into a glass container with a lid. Next, 1 mL of a solution containing 2.5% H 2 SO 4 and 98% methanol (1:40 v/v) was added to each container and then put at 80 °C for 1 h. To extract fatty acid methyl ester, first samples were cooled to room temperature, then, 500 μL of hexane was mixed with 1.5 mL Survival rate = (The number of larvae remained at the end of the period ∕The number of larvae stored at the beginning of the period) × 100 of 9% NaCl and added to the sample. Following centrifuging of the sample (10 min at 4000 rpm), supernatant (containing hexane) was removed and injected into the gas chromatography (GC) system to determine the fatty acid composition. A gas chromatography machine (Model 4600-. Unicam Company, England) was utilized for the separation and determination of fatty acids. The column dimensions of this device were as follows: Bp10, 30 m × 0.1 mm. detector: FID, carrier gas: helium at 30 mL s −1 , oxygen at 300 mL s −1 , detector temperature: 250 °C, injector temperature: 240 °C and column temperature: 200 °C. Using a Hamilton syringe, 1 µL of the sample of interest was injected into the system. Due to the passing through the helium gas and gradual heating, the methyl esters of fatty acids turned into vapor, left the column successively, and a curve was drawn, and then, the retention time of each fatty acid was specified. The retention time of each fatty acid was compared with the standard curve and finally, the type and quantity of fatty acids were determined (according to % of total fatty acids) (Pal et al. 2013).

Amino acid composition
To assess the amino acid profile, the procedure developed by Lindroth and Mopper (1979) was used with minor modifications. Initially, 0.1 g of dried shrimp from each replicate was added to the digestion tube in a freeze dryer (Model: FDU-7012, Operon, South Korea) and then 7.5 mL of 6.00 N hydrochloric acid (HCl) was added, and after removing the air inside the tube with nitrogen gas, it was put into the oven at 110 °C for 24 h. Next, the acid in the tube was diluted with distilled water to 25 mL and the solution was filtered with syringe filters, 0.45 µm, and 10 µL filtered solution was poured into glass containers. The samples were put under vacuum conditions, and eventually, it was stored in the fridge. After digestion, for separation, 10 µL acetate buffer was added to the digestion tube containing dried amino acid, and after mixing, again 490 µL acetate buffer was added to the mixture and incubated for 5 min. Following that, borate buffer and 100 µL of OPA (o-phthaldialdehyde) solution were added and after 2 min of incubation, 50 µL of 0.75 M hydrochloric acid was added to the reaction mixture until the reaction was stopped. Ultimately 20 µL of the final mixture was injected into the column (18RP mm OPA specific column 100 × 4, column temperature = 30 °C) by HPLC device syringes (1290 infinity of England).

Sterol extraction and measurement
To extract free sterols, 20 mL dichloromethane (DCM) was added to 1 g of dried shrimp powder and thoroughly mixed and homogenized by using an ultrasonic homogenizer. The mixture was kept still for half an hour at room temperature and filtered using Whatman™ qualitative filter paper, grade 1, and finally, the solvent was completely removed via vacuum evaporation and nitrogen gas. Following the extraction of free sterols from four types of mollusks, the extracts were dried using nitrogen gas. Next, 50 µL of double distilled dry pyridine and 50 µL of BSTFA reagent (N,O-Bis(trimethylsilyl)trifluoroacetamide > 99%) containing 1% TMCS (trimethylchlorosilane) (Sigma-Aldrich Co.) were added. The samples were then stored overnight in the dark at room temperature. Diluted with 1 mL of dichloromethane, 1 µL of each sample was injected into the gas chromatography analytical instrument for further analysis. Each extraction procedure was carried out three times. A gas chromatography machine (4600-. Unicam Company, England) was employed for the purification of sterols in the extracts. The column specifications are as follows: Bp10, 30 m × 0.1 mm. detector: FID, carrier gas: helium filled at 6 Pa. The thermal program of the column is as follows: starting at 150 °C for 2 min, it reached 300 °C by increasing the temperature by 5 °C per minute and remained at the same temperature for 15 min. The temperature of the detector, as well as the injection chamber was 300 °C and the injection volume was 1 µL. Identification of the sample components in the chromatogram was performed by using retention time. The retention time of different components was measured by taking advantage of the data obtained from the injection of phytosterol standards (campesterol, stigmasterol, and β-sitosterol) (Sigma-Aldrich Company) under the same conditions as the injection of samples, and cholesterol was used as an internal standard for quantitative analysis (Liu et al. 2007).

Shrimp homogenization
To assess the content of total phenolics, flavonoids, and antioxidant activity, a total of 10 shrimps were selected from each replicate. They were then homogenized in a 1:10 (w/v) ratio in phosphate buffer solution (PBS) containing 8 g NaCl, 0.2 g KCl, 1.42 g Na 2 HPO 4 , and 0.24 g KH 2 PO 4 , pH7.2. Subsequently, the supernatant fraction obtained from the homogenized samples centrifuged for 10 min at 4 °C (1500 rpm) was employed for the measurement of total phenolics, flavonoids, and antioxidant activity (Akbary et al. 2021c).

Phenolics content measurement
The total phenolic contents, in all the samples, were calculated using the method developed by Ebrahimzadeh et al. (2008) with slight modifications. A total of 200 µg of shrimp extracts were mixed with 20 µL Folin-Ciocalteu reagent (diluted with distilled water in a 1:10 ratio) and were left at room temperature for 4 h. Then 2 µL of 6% sodium bicarbonate was added and mixed, and after 15 min at room temperature, optical absorption was detected using a Shimadzu UV-1800 spectrophotometer (Shimadzu, UV-1800, Japan) at 765 nm. Total phenolics were measured using a standard curve and total phenolic content was expressed in terms of gallic acid equivalent (mg of GAE g −1 of extract).

Flavonoids content measurement
Flavonoid content was estimated using the method developed by Ebrahimzadeh et al. (2008) with slight modifications. Briefly, 0.5 mL of each extract was mixed with 1.5 mL of 95% methanol, 0.1 mL of 10% aluminum chloride hexahydrate (AlCl 3 ), and 0.1 mL of 1 M potassium acetate (CH 3 CO 2 K), and 2.8 mL distilled water. After 30 min at room temperature, optical absorption was read on a Shimadzu UV-1800 spectrophotometer at 415 nm and calculated using a standard curve of quercetin and expressed as mg of quercetin equivalent (mg QE g −1 ) per gram of dry sample.

Antioxidant activity measurement
The reaction mixture to assay superoxide dismutase (SOD) consists of HEPES-KOH pH7.8 containing 0.1 mM EDTA, 50 mM sodium carbonate buffer, pH7.2, 12.7 mM metallothionein, 75 μM nitro blue tetrazolium, 1 μM riboflavin, and 200 µL of the extracts. Exposed to 15 min light, the absorbance of the samples at 560 nm was read using a spectrophotometer, and a test tube containing all reaction mixture components except the enzyme extract was utilized as a control (blank) (Winterbourn et al. 1975).
Catalase (CAT) activity assessment was carried out according to hydrogen peroxide analysis with a decrease in light absorbance at 240 nm for 30 s and expressed per mg of protein in the enzyme extract, in which, 20 mM sodium phosphate buffer, pH7, and 20 μL of 30% hydrogen peroxide were employed as electron acceptors (Dazy et al. 2008).
Glutathione peroxidase (GPX) activity was determined according to the protocol developed by Lawrence and Burk (1976). Briefly, 0.9 mL of the reaction mixture containing 50 mM phosphate buffer pH7.0, 1 mM EDTA, 1 mM sodium azide (catalase inhibitor), 0.2 mM β-NADPH, 20 μM glutathione reductase, and 1 mM GSH was incubated for 15 min at 25 °C. Then, 0.1 mL of 0.25 mM hydrogen peroxide and 50 µL of enzyme extract were added to the mixture, and light absorbance was detected at 440 nm by using a spectrophotometer.
Malondialdehyde (MDA) assay was carried out according to the procedure developed by Baluchnejadmojarad et al. (2010). Initially, 375 mg of thiobarbituric acid (TBA) was dissolved in 2 mL HCl, mixed with 15% (100 mL) trichloroacetic acid (TCA), and then they were kept at a hot water bath at 50 °C to completely dissolve sediment particles. Next, 1 mL of the extract was mixed with 2 mL of this solution and heated for 45 min in boiling water until it turned orange. Following that, the samples were centrifuged for 10 min at 1000 rpm, and at the end, the absorbance was read at 535 nm.

Statistical analysis
Data analysis was conducted via one-way ANOVA and comparing treatment means by applying Duncan's multiple range test at a significance level of 0.05. The investigation of the normality of the data was conducted by applying the Kolmogorov-Smirnov normality test. Besides, Levene's test was used to assess the equality of variances, and all data were analyzed utilizing the statistical software package of SPSS (SPSS Inc., version 19, Chicago, IL, USA), and Excel 2010 software. Table 3 presents the growth performance and feed consumption for L. vannamei shrimps fed with various levels of MPE following the 8-week experiment period. At the end of the feeding trial, the final weight (FW) of the control shrimp was 548.66 mg while the FW of the groups treated with MPE ranged between 777.33 ± 26/08 and 1234 ± 50.10 mg. The growth performance data (Table 3) showed the dietary supplementation with MPE at 15 g kg −1 diet resulted in significant increases in the SGR, LW, WG, and PER compared to the other MPE groups and the control group. Whereas the lowest those values were recorded for the MPE 0 group. The FCR in the group fed with MPE 15 was lower than the shrimps on other experimental diets (P < 0.05). However, the survival rate obtained with experimental diets did not show a significant difference (P < 0.05).

Chemical composition
The body chemical composition of shrimp fed with experimental diets containing specific levels of MPE is presented in Table 4. The highest level of crude protein was reported in the MPE 10 and MPE 15 groups, which showed a significant difference from the MPE 5 and MPE 0 groups (P < 0.05). No significant difference was observed between the levels of crude fat and dry matter in shrimps fed with MPE 5 and MPE 10 (P < 0.05), while the highest levels of fat and dry matter were recorded for the MPE 15 group. There was also no significant difference among the groups on the experimental diets with regard to the amount of ash (P < 0.05).

Amino acid composition
The total amino acid composition of L.vannamei shrimp fed with experimental diets containing a specific amount of MPE is indicated in Table 6. As a result of the inclusion of MPE in the diet, the mean total body amino acids of shrimp increased as compared with the shrimp on the control diet (PME 0 ). The amount of amino acid composition rose with an increase in the concentration of MPE in the shrimp diet. The highest average total essential amino acid (4.12 ± 0.05 g AA 100 g −1 ), non-essential amino acid (4.30 ± 0.02 g AA 100 g −1 ), and total amino acid (± 0.02 8.32 g AA 100 g −1 ) levels were reported for the MPE 15 group, reflecting a significant difference from other diets (P < 0.05).

Sterols
The comparison of total free sterols and total sterols in the body of L. vannamei shrimp fed with MPE experimental diets is presented in Table 7. Sitostanol was the predominant sterol among groups fed with MPE (Fig. 1). Cholesterol was the only free sterol in shrimp fed MPE 0 . However, the highest levels of free sterols and total sterols were observed in shrimps fed with MPE 15 , demonstrating a significant difference from other diets (P < 0.05).   Fig. 1 Chromatogram of the main composition of sitostanol in Litopenaeus vannamei shrimp fed with premix extract (MPE) of brown Sargassum ilicifolium, Nizimuddinia zanardini, Cystoseira indica, and Padina australis macroalgae MPE 15 (3.40 ± 0.16 mg GAE g −1 dry extract), reflecting a significant difference from the MPE 10 (2.4 ± 0.2 mg GAE g −1 dry extract) and MPE 5 (1.50 ± 0.1 mg GAE g −1 dry extract) (P < 0.05) groups. Moreover, this difference was reported to be significant between MPE5 and MPE10 (P < 0.05). The highest flavonoid content (0.09 ± 0.0006 mg QE g −1 sample) was observed in the MPE 15 group (P < 0.05), showing a significant difference from the other groups (P < 0.05). A significant difference was also observed between MPE 5 and MPE 10 (P < 0.05).  Table 8 presents the variations of the antioxidant state in the shrimps fed with MPE. There was no significant difference between the groups fed with MPE and the control group (MPE 0 ) regarding the levels of SOD (P < 0.05). On the contrary, GPX and CAT activity significantly in all the MPE-treated groups compared to the control. The highest GPX (90.33 ± 1.52 U mL −1 ) and CAT (102.66 ± 2.08 U mL −1 ) were shown in MPE 15 group. The lowest MDA content (1.56 ± 0.05 nmol min −1 mg −1 protein) was reported in shrimps fed with MPE 10 and MPE 15 , showing a significant difference from the group fed with MPE5 and MPE 0 (P < 0.05).

Discussion
According to the results of this study, adding MPE to the diet improved the growth of the shrimps, and the highest specific growth factor, weight gain and protein efficiency were recorded for the group fed with MPE 15 . Similar results were also reported for L. vannamei with 1 g P. australis polysaccharide extract kg −1 feed (Akbary and Aminikhoei 2018c). Likewise, to the best of our knowledge, there was no study on the use of the macroalgae premix extract in shrimp diet prior to this research. However, the inclusion of algae and algae extracts in the diets of shrimps has garnered considerable attention due to its reported high levels of protein and many other growth stimulants. Hence, it can improve growth and appetite, stimulate immunity and reinforce resistance to pathogens (Schiener et al. 2014;Akbary and Aminikhoei 2018a;Akbary et al. 2021a), which are in line with the findings of this study and indicates that algal polysaccharide can improve the growth of beneficial bacteria and intestinal health and also stimulate shrimp growth. Moreover, the improvement in shrimp growth can be attributed to the presence of active substances such as flavonoids and tannins in the diet containing the aforesaid macroalgae, which increased feed consumption. One of the positive mechanisms of macroalgae that influences growth is the presence of compounds such as amino acids and essential fatty acids, which are substantially involved in the growth and vital functions of shrimps (Akbary et al. 2021a). The lowest levels of the FCR were observed in white shrimps fed with 1 g P. australis algae extract kg −1 feed (Akbary and Aminikhoei 2018c) and 1.5 g J. adhaerens red algae extract kg −1 feed , which is in line with the findings from this research. The results regarding the protein efficiency rate after the inclusion of algae in white shrimp feed were positive (Cruz-Suárez et al. 2009;, which complies with the results of the present study. The improvement in the consumption of protein may vary by algae species. For instance, the PER of shrimps fed with a diet containing Ulva was higher than shrimps on the diet containing Macrocystis and Ascophyllum algae (Cruz-Suárez et al. 2009). However, it is assumed that algae can increase the absorption and digestion of dietary proteins or regulate lipid metabolism ).
According to the results of this study, the inclusion of MPE in the shrimps' diet improved the carcass quality. The highest level of crude protein was recorded for the group fed with MPE 10 and MPE 15 , while the highest level of fat and dry matter was recorded in the MPE 15 group. Choi et al. (2015) revealed that adding 20 g kg −1 of red algae extract (P. yezoensis) significantly increased the crude fat content in the olive flounder as compared with the control group, but there was no significant difference between different concentrations of red algae extract, which is consistent with the results of this study. It could also be argued that protein in the metabolism process takes its primary path, i.e., the tissue synthesis path, due to the inclusion of algae extract in the feed and is stored in the form of protein (Shalaby et al. 2006). Besides, the use of algae extract in the diet plays a major role in fat synthesis and metabolism. Also, this study was consistent with studies involving other aquatic species. Nakagawa et al. (1997) demonstrated that the use of Ulva algae powder changed the fat metabolism in the blackhead seabream, Acanthopagrus schlegelii and the use of algae powder led to the storage of body fat and reduced body weight loss during winter. Ergün et al. (2008) studied the effect of sea lettuce extract, Ulva on the body chemical composition of nile tilapia fish, Orechromis niloticus and showed that fish fed with 5% of algae extract contained less carcass fat than the control group. It could be stated that the inconsistency of results originates from the difference in the method of using algae (algae powder and extract), the difference in the study fish species, the duration of algae use, the algae type, and the difference in the experiment environmental conditions.
The amount of PUFA in the groups fed with MPE was significantly higher than the control group, while the highest level of PUFA was observed in the MPE 15 group, which complied with the study conducted by Choi et al. (2015), who indicated that saturated fatty acids are substantially involved in increasing the PUFA level in the muscles of fish fed with algae extract (Choi et al. 2015). Choi et al. (2014) showed that the inclusion of Hizikia fusiformis algae glycoprotein in the diet of olive flounder, Paralichthys olivaceus, led to changes in PUFA levels, including DHA, ARA, linoleic acid (LIA), and EPA (Choi et al. 2014). Choi et al. (2015) suggested that the use of 20 g kg −1 feed of red algae Pyropia yezoensis increased DHA, ARA, and LIA in the muscle of olive flounder, which is in line with the findings from this study. The main reason for the change in lipid metabolism following the addition of algae to the diet yet is still unknown, but the results of relevant research indicates that the addition of seaweed to diet leads to a positive change in the process of lipid metabolism, thereby increasing the amount of PUFA and the positive efficiency of stored lipids. In addition, foods high in protein and carbohydrates are likely to cause excessive satisfaction of energy within the aquatic species, which is converted to fatty acids and stored as lipids (Choi et al. 2014). Similar to these results, Akbary et al. (2021a) investigated the fatty acid composition in the muscle of L. vannamei fed with the extract of I. stellate brown algae and reported that the highest levels of PUFA and EPA were observed in shrimps fed with 1 and 1.5 g algae extract kg −1 feed. It could be stated that the premix extract of the study macroalgae is a great source of ω3 fatty acids (e.g., DHA and EPA) and is capable of increasing the muscle lipid profile in proportion to the ratio of ω3 fatty acid concentration (Güroy et al. 2007;) which reduces the 3-n/6-n ratio. This finding is in line with the study by Choi et al. (2015).
The amino acid compound in many macroalgae can be considered relatively complete regarding the essential amino acids. Many species of algae contain the majority of essential and non-essential amino acids (Ortiz et al. 2006;Gressler et al. 2010). Based on the results of this study, as the concentration of MPE added to the diet grows, the total essential amino acids, non-essential amino acids, and total amino acids in the body of shrimps, L.vannamei, increased. Kalaiselvi et al. (2018) reported that Artemia nauplii enriched with ether extract, acetone extract, and especially ethanolic extract of Phyllanthus amarus significantly increased the total protein level and amino acid and lipid concentration of freshwater shrimp larvae, Macrobrachium rosenbergii, as compared with non-enriched Artemia nauplii. Moreover, diets containing U. lactuca and G. vermiculophylla promoted amino acid digestibility in L. vannamei (Anaya-Rosas et al. 2019) which is consistent with the results of this study. The amino acid composition of algae has been investigated numerous times. In this study, glutamic acid accounted for 21.35% of total amino acids. In most brown algae, glutamic acid accounts for between 22 and 44% of total amino acids (Øverland et al. 2019), which complies with the findings from this research. It can be said that the imbalance of amino acids in the diet leads to an increase in the oxidation of amino acids and, as a result, a decrease in the growth and conversion efficiency of fish. Therefore, the best way to compensate for the lack of essential amino acids is to use macroalgae containing essential amino acids (Rajapakse and Kim 2011).
Sitostanol was the major sterol in shrimps fed with MPE, while cholesterol was the only free sterol in MPE 0 . Cholesterol forms the central core of steroids. Other researchers have specified in their reports that cholesterol is the main steroid in red algae, and its amount is significantly higher than that of brown algae (Saeidnia et al. 2012;Akbary et al. 2021b). However, this compound was observed in brown algae such as Padina australis and S. marginatum (Akbary et al. 2021b), which is in line with the results of the present study. It could be stated that the type of these compounds varies by species in environmental conditions. These compounds are among the secondary metabolites and the secondary metabolites of any living organism vary or transform into similar derivatives under environmental conditions (Desmond and Gribaldo 2009). Given the considerable medicinal value of sterols, including campesterol, stigma sterols can serve as suitable sources in the pharmaceutical industry, where these sterols play a substantial role in preventing the growth of cancer cells and cardiovascular diseases (Fernandes and Cabral 2007).
The antioxidant property grows with an increase in the content of the phenolic and flavonoid compounds. Flavonoids and tannins are highly capable of removing free radicals, and this capability is mainly determined by the number of aromatic rings and the nature of the moving hydroxyl groups (Akbary et al. 2021c). The results of this study mirrored the direct link between an increase in MPE concentration and the phenol and flavonoid content, while the phenol and flavonoid content in shrimps fed with MPE 0 was zero. According to the results of this study, the phenol and flavonoid content of the premix aqueous extract of the studied brown macroalgae included 83.46 ± 7.07 mg of GAE g −1 extract and 10.01 ± 0.98 mg QE g −1 dry extract. After assessing the total phenol content and antioxidant properties in three macroalgae species from Chabahar coast in Iran, Akbary et al. (2021c) reported that the total phenol content in the aqueous extract of certain species of brown algae, namely P. australis (69.66 ± 2.08 mg GAE g −1 extract), S. marginatum (72.33 ± 2.08 mg GAE g −1 extract), and Ahnfeltiopsis pygmaea (76 ± 2 mg GAE g −1 extract), indicated that the phenol content in the aqueous extract of the three macroalgae species was lower than the macroalgae premix as compared with the present study. It could be stated that different macroalgae species contain different amounts of phenolic and flavonoid compounds (Hongayo et al. 2012). Wood and Enser (1997) indicated that diets containing antioxidants can enhance the fatty acid profile of meat. This may be additional reasons for the enhancement of the fatty acids content recorded in the current study because one of the important ingredients of algae is phenolic compounds which are responsible for antioxidant activity (Hongayo et al. 2012). For instance, Tenorio-Rodriguez et al. (2017) reported that among the 17 large algae including green, red, and brown algae, the brown macroalgae extract was found to have the highest antioxidant activity and sources of natural bioactive compounds, and the antioxidant activities of algae can be attributed to the presence of various secondary metabolites such as phenolic compounds and carotenoids (Hongayo et al. 2012). Besides, phenolic compounds of macroalgae are of importance as potential factors in improving the health and performance of aquatic animals (Naiel et al. 2020(Naiel et al. , 2021. Also, these compounds are typically used to prevent lipid per oxidation and increase shelf life (Holdt and Kraan 2011).
According to the results of this study, the inclusion of MPE in the diet improved the antioxidant activity of the L. vannamei shrimp as compared with shrimps on the control diet. Akbary and Aminikhoei (2018a) reported that the inclusion of 1.5 g kg −1 of U. rigida algae extract in the diet of shrimps significantly increased the activity of SOD and reduced glutathione. Akbary et al. (2021a) also indicated that 1 g concentration of I. stellate increased the level of SOD (19.32 U mg −1 protein), GPX (249.06 U mg −1 of protein), CAT (31.19 U mg −1 of protein), and phenol oxidase (31.19 U mg −1 of protein), which is seemingly in line with the results of this research. It can be stated that antioxidant potential of algae by various mechanisms, such as reducing peroxide radicals and converting them into oxygen and water, has been confirmed in previous studies (Akbary et al., 2021a). Algae contain active biological compounds (polyphenols, glycosides, anthocyanin, tannins, and thiocarbamates). These active compounds expel free radicals and active antioxidant enzymes and inhibit oxidases (Shi et al. 2005). Concentration of MDA is indicative of toxic processes triggered by free radicals, and the level of MDA serves as a good indicator of the level of lipid peroxidation (Peixoto et al. 2016). Based on the results of this study, the lowest MDA level was observed in MPE 15 , which complies with the research by Peixoto et al. (2016) and Akbary et al. (2021a).

Conclusion
In general, the results of this study revealed that the premix extract of brown Sargassum ilicifolium, Nizimuddinia zanardini, Cystoseira indica, and Padina australis macroalgae improved the growth performance, chemical quality, amino acids and omega-3 unsaturated fatty acids quantity, and sterol, phenol, and flavonoid contents and increased the antioxidant activity of L. vannamei shrimps. Hence, the inclusion of 15 g kg −1 of the premix aqueous extract of the aforementioned brown macroalgae, as growth and antioxidant status in the diet of L. vannamei shrimps is recommended.