Influence of stocking density on the growth, immune and physiological responses, and cultivation environment of white-leg shrimp (Litopenaeus vannamei) in biofloc systems

Biofloc (BF) stands out as a promising system for sustainable shrimp farming. Optimizing various culture conditions, such as stocking density, carbohydrate source, and feeding management, is crucial for the widespread adoption of the BF system. This study compares the growth performance of white-leg shrimp (Litopenaeus vannamei) in culture ponds at low density (LD) with 50 organisms/m2 and high density (HD) with 200 organisms/m2. Post-larvae of white-leg shrimp were stocked for 16 weeks in both LD and HD groups. The LD group exhibited a superior survival rate, growth rate, and feed consumption compared to the HD group. The BF from the LD system recorded a significantly higher protein content (16.63 ± 0.21%) than the HD group (15.21 ± 0.34%). Heterotrophic bacterial counts in water did not significantly differ with stocking density. However, Vibrio count in water samples was higher in the HD group (3.59 ± 0.35 log CFU/mL) compared to the LD group (2.45 ± 0.43 log CFU/mL). The whole shrimp body analysis revealed significantly higher protein and lipid content in the LD group. In contrast, the total aerobic bacterial count in shrimp from the HD group was high, with the identification of Salmonella enterica ssp. arizonae. Additionally, Vibrio counts in shrimp samples were significantly higher in the HD group (4.63 ± 0.32 log CFU/g) compared to the LD group (3.57 ± 0.22 log CFU/g). The expression levels of immune-associated genes, including prophenoloxidase, transglutaminase, penaiedin 3, superoxide dismutase, lysozyme, serine proteinase, and the growth-related gene ras-related protein (rap-2a), were significantly enhanced in the LD group. Conversely, stress-related gene expression increased significantly in the HD group. Hepatopancreases amylase, lipase, and protease were higher in the LD group, while trypsin activity did not differ significantly. Antioxidant enzyme activity (catalase, glutathione, glutathione peroxidase, and superoxide dismutase) significantly increased in the LD group. The histological structure of hepatopancreas, musculature, and female gonads remained similar in both densities. However, negative effects were observed in the gills' histology of the HD group. These results suggest that increasing stocking density is associated with significantly negative biological, microbial, and physiological effects on white-leg shrimp under the BF system.


Shrimp farming system
The experiment was conducted from July to October 2021, spanning 16 weeks, at a commercial shrimp hatchery in Damietta, Egypt.Six cement ponds, each with a total capacity of 36 m 3 , were utilized.Each pond measured 3 m in width, 10 m in length, and 1.2 m in depth.Sand-filtered seawater (32 ± 0.5 ppt) from the Mediterranean Sea, totaling 30 m 3 , was introduced into each pond.To maintain optimal conditions, the experimental units received continuous aeration and were subjected to a 12-h dark/12-h light cycle.Central air pumps (12 distributors/ pond, 5.5 horsepower, Turo Vortex Pumps ® ) facilitated aeration.The aeration line, consisting of 2-inch pipes, was divided into units with flow regulators to fine-tune air compression in all ponds.A network of disk diffusers (each measuring 26 cm) was securely fixed and installed at the bottom of each pond to ensure effective aeration and thorough mixing of the culture water across all ponds 4 .

Experimental groups and biofloc setup
Post-larvae of white-leg shrimp (L.vannamei) with a mean body weight of 0.02 ± 0.001 g were stocked at densities of 50 and 200 organisms/m 2 , with triplicates for each treatment.To establish the BF, we followed the method outlined by Said et al. 4 .Wheat flour, containing 58.3% carbon, was introduced once daily to all experiments as a carbohydrate source to stimulate the development of the BF community 26 .Wheat flour was chosen due to its efficient utilization and positive impact on water quality parameters when used in BF systems 27,28 .The carbonto-nitrogen (C:N) ratio was maintained at 15:1 by adding the necessary amount of the carbon source.The preweighed carbon supplement was mixed with pond water and evenly distributed across the surfaces of the ponds.

Management of experimental units
Post-larvae were fed four times a day at 7:00 AM, 11:00 AM, 2:00 PM, and 5:00 PM with a commercial shrimp feed (Skretting ® , El-Sharkia, Egypt) containing 38% crude protein.The proximate analyses of both the feed and carbon bases used in this study are detailed in Table 1.The post-larvae were fed daily as a percentage of the total biomass, with daily feeding rates gradually decreasing from 32 to 1.9% over the experimental period.
Feed quantities were adjusted every two weeks based on Van Wyk 29 , taking into account any observed mortality.Monitoring of temperature, dissolved oxygen, nitrite, ammonia, BF volume, and turbidity was conducted

Macronutrient analysis of shrimp and biofloc
The composition of BF communities and shrimp whole bodies was assessed following the guidelines of the Association of Official Analytical Chemists 31 .At the conclusion of the experiment, shrimp samples were collected from each pond.BF masses were collected using a 100 mm mesh net for subsequent biochemical analysis.These samples were then dehydrated in a Nabertherm ® heating oven at 60 °C, ground, and subjected to biochemical analysis.
Moisture content was determined by drying a fixed quantity of samples (5 g) at 105 °C in a binder heated oven until a consistent weight was achieved.Ash content was estimated by burning a fixed number of dried samples in a muffle furnace at 550 °C for 4 h, followed by cooling and weighing.
Crude proteins were quantified using the Kjeldahl method (FOSS ® , KjelTec ™ 84,000).Fat extraction was performed using an automated fat extraction system (FOSS ® , Soxtec ™ 8000).The estimation of crude fiber employed an automatic fiber analyzer system (FOSS ® , Fibertec ™ 8000), while the nitrogen-free extract was calculated by taking the difference.

Bacterial analysis
At the conclusion of the experiment, ten randomly selected shrimp and 100 mL water samples were collected from each pond.These samples were transported aseptically to the fish microbiology laboratory at the Fish Resources Faculty, Suez University, and were examined immediately.
Shrimp samples were prepared, and heterotrophic and Vibrio bacterial counts were estimated following the FDA guidelines 32 .Colonies of the selected bacteria underwent Gram staining and were identified biochemically using indole, Voges-Proskauer, and methyl red tests.Suspected Vibrio colonies were subjected to oxidase biochemical tests and API 20E diagnostic strips 33,34 .Bacterial species were then confirmed using API 20E strips purchased from BioMérieux, France 35,36 .

Expression analysis of immune-, stress-, and growth-related genes
At the conclusion of the experiment, three organisms from each group underwent hemolymph sampling for total RNA extraction using TRIzol reagent (QIAzol ® , QIAGEN).Synthesis of cDNA from 1.5 μg of RNA was accomplished using the Revert-Aid First Strand cDNA Synthesis Kit ® (Thermo Fisher Scientific).
Gene expression was calculated using the β-actin gene as an internal reference.Expression results were presented as fold change using the 2 −ΔΔCT method according to Livak and Schmittgen 37 replacing control values with higher density values.Data are present as fold changes compared to higher density (200 organisms/m 2 ).

Digestive enzymes
The pooled hepatopancreases of shrimp were weighed, homogenized with sterilized distilled water, balanced, and then uniformly mixed with chilled phosphate buffer (0.65%, 1:10 w/v, pH 7).The resulting supernatants  38 , known as the '3, 5-dinitrosalicylic acid' method.Amylase activity was determined from the maltose standard curve, with the amount of maltose obtained from starch/min/mg protein at 37 °C.Protease activity was measured according to Drapeau 39 using the casein digestion method.Tyrosine release was estimated from the standard curve, indicating one micromole of tyrosine as unit/mg protein at 37 °C.Lipase enzyme activity was determined by the milliequivalents of alkali consumed.Trypsin enzyme activity was assessed following the technique of Zhang et al. 40 with casein substrate.One unit of trypsin was defined as the number of micromoles of tyrosine freed per minute per milligram of protein.

Antioxidant enzymes
About 200 μL of pooled hemolymph was withdrawn from the ventral sinus of a shrimp, located at the bottom of the primary abdominal segment.This hemolymph was then transferred into a 1 mL sterilized syringe and mixed with an anticoagulant mixture.The mixture comprised 0.34 M sodium chloride, 30 mM trisodium citrate, and 10 mM EDTA-Na 2 , as detailed by Xu and Pan 41 .The resulting mix was adjusted to a pH of 7.55 and an osmolality of 780 mOsm kg −1 .
Next, the anticoagulant-hemolymph mix from 10 shrimp in each pond was pooled, gently mixed, and subjected to centrifugation at 800 × g for 10 min at 4 °C.The supernatant obtained after centrifugation was withdrawn as plasma samples and stored at − 80 °C for subsequent antioxidant analysis.
For the assessment of superoxide dismutase (SOD) activity, the "hydroxylamine method" outlined by Xu and Pan 42 was employed.Catalase activity was determined using the "visible light method" as described by Kim et al. 43 , while the "turbidimetric method" specified by Feng et al. 44 was utilized for measuring glutathione peroxidase activity.

Histological examination
Thirty samples, comprising 15 from each group, were collected.The gills, intestine, stomach, hepatopancreas, and female gonad were promptly excised and fixed in a 10% formalin solution for 24 h.Subsequently, the samples underwent a thorough process, including washing with distilled water, dehydration with a series of ethyl alcohol concentrations (70%, 80%, 90%, 95%, and 100%), overnight immersion in methyl benzoate, and two rounds of clearing with xylene.Following these preparations, the samples were fixed in 65-70 °C paraffin, sliced into sections of 5-6 μm, and stained with hematoxylin/eosin.The examination of the samples was conducted using a Leica ® ICC50 HD light microscope.

Statistical analysis
The impact of various stocking densities on different parameters was analyzed using the t-test in IBM SPSS Statistics version 26 (IBM Corporation, NY, USA).Results were presented as mean ± SE, and differences in means were assessed using Duncan's multiple-range test.A probability value of < 0.05 was employed to signify statistically significant differences.www.nature.com/scientificreports/

Ethical approval
The study has been approved by the Faculty of Fish Resources, Suez University, under the protocol of international and national guidelines for the ethical treatment of animals.

Results and discussion
Water quality, growth performance, survival rate, and feed utilization All measured water quality parameters, including dissolved oxygen (DO), pH, NH 3 , NO 2 , and turbidity, did not exceed the appropriate levels for white-leg shrimp culture 45 in both groups throughout the entire experimental period (Table 3).Significant differences in growth-performance parameters and survival rates were observed between the two densities (Table 4).The final individual weight increased by 5.66 g in the lower density (LD) compared to the higher density (HD).Similarly, specific growth rates (SGR%) showed a significant increase of 0.32%/day in the LD group.Weekly growth was significantly enhanced in the LD group, with a biomass weight gain percentage showing a significant increase of 292.72 compared to HD. Notably, the survival rate in the LD group was approximately 99%, compared to 97% in the HD group.In contrast, total biomass was higher in the HD group than in the LD group (Table 4).
Acceptable growth performance and high survival rates were achieved in both systems, even at higher density, in agreement with El-Sayed 46 .The BF system demonstrated greater holding capacity than conventional clear water aquaculture 28 .The high survivability and growth in the BFT systems may be attributed to favorable environmental conditions, coupled with the high nutritional value of flocs for shrimp.Additionally, microbial communities within the flocs could enhance the activity of digestive enzymes, gastrointestinal microflora, and overall nutrient utilization 41 .Successful super-intensive culture in BFT systems for rearing white-leg shrimp at HD reflects the species' ability to achieve better growth performance and higher survival 47 .
The current results revealed that the higher stocking density treatment led to lower growth performance and survival compared to the lower density.A negative relationship between stocking density and shrimp growth had been previously documented 11,[48][49][50] .This could be attributed to the energy expended on food competition and the stress caused by a larger number of animals 13,51 .Fleckenstein et al. 47 reported that higher final weight and faster growth rates were achieved in LD treatments (100 shrimp/m 3 ) compared with 200 shrimp/m 3 .Additionally, LD showed slightly higher survival (94.5%) compared to HD (91.3%).
The results of the current study showed a significantly higher total biomass in the higher stocking density treatment, aligning with Fleckenstein et al. 47 .These authors reported significantly superior total shrimp biomass production in the HD group (4.0 kg/m 3 ) compared to the LD group (2.3 kg/m 3 ).The notable increase in biomass production in high-density stocking rates could provide a solid rationale for using elevated shrimp densities.
In the current study, improved feed efficiency (FE) was observed in the LD group (50 shrimp/m 2 ) compared to the HD (200 organisms/m 2 ).Additionally, the protein efficiency ratio (PER) increased by 0.24 in the LD group compared to the HD group (Table 4).Fleckenstein et al. 47 reported low FCR values, ranging from 1.0 to 1.1 in low-density (2.3 kg/m 3 ) BF treatments.The enhanced feed utilization in BF systems may be attributed to indications that the presence of BF communities could elevate enzymatic activity in the digestive canal.Consequently, this increase in enzymatic activity is contributing to improved feed efficiency and reduced FCRs 52 .
Vungarala et al. 12 and Nguyen et al. 53 noted higher FCR values with increased stocking densities of white-leg shrimp.The observed better feed utilization parameters in the lower density system indicate a reduced ability of shrimp to graze on the flocs at high density 24 .The higher PER value in the LD system could be explained by the sustained availability of protein-rich BF particles, which can be consumed by the shrimp, and/or the stressful conditions in the high-density system (crowding, less favorable water quality, etc.) 54,55 .

Analysis of macronutrients in biofloc and shrimps
A proximate analysis of BF composition taken from the LD group revealed a numerical superiority in lipid content (Table 5).Additionally, significantly higher protein and carbohydrate contents were noted in the LD group, while ash and fiber contents from the HD system (200 organisms/m 2 ) were both higher than those in the LD system (50 organisms/m 2 ) (Table 5).
BF particles possess good nutritional value and can be used as aquatic food along with artificial feed 56 .The results of the present study demonstrated an enhanced nutritive value of the BF in the LD system.Various proteins, lipids, and ash contents in the BF particles were found in previous studies.Tao et al. 57 found that the average protein content in floc particles gathered at different stocking densities varied from 28.9 to 29.2%.Khoa et al. 58 recorded that protein levels in floc particles ranged from 22.2 to 25%.Regarding lipid content, Tao et al. 57 found that lipid contents in floc particles ranged from 5.4 to 5.7%.Additionally, different ash content in flocs was reported; for example, Tao et al. 57 found that ash contents were approximately 29.1% without differences in all stocking densities, and these differences might be due to the diversity in carbon sources applied 59 .
The nutritional value of BF may vary between treatments due to changes in the microbial community.Floc size can also influence its nutritional composition; Ekasari et al. 10 and Ray et al. 60 reported increased protein and lipid content in floc with an increase in floc particle size.Additionally, C:N ratios can modify the microbial and nutritional composition of BF.Panigrahi et al. 61 reported that the C:N ratios (5; 10; 15; 20) had a significant impact on the developmental growth and characteristics of BF.
The whole-body proximate composition of shrimp showed significantly higher protein and lipid content in the LD compared to the HD treatment (Table 5).Similar to the BF analysis, significant ash content was found in shrimps of the HD system.However, the two treatments were not significantly different in fiber and ash contents (Table 5).
High contents of crude proteins, lipids, and ash were observed in the shrimp cultured in the BFT.Rajkumar et al. 28 recorded that using carbohydrates in the BFT caused a surge in protein utilization efficiency and supported essential lipids and vitamins for the shrimp.The change in the body composition of the shrimp may be attributed to various factors such as water quality, stress, nutrient abundance, and feed intake and utilization 62 .The decreased lipid content in the HD system compared to the LD system can be a result of reduced feed intake and low amounts of accumulated lipids in the carcass structure 63 .However, another study reported that different densities had no significant differences in the high content of crude protein, lipid, and ash in the shrimp in the BFT 12 .Also, no variations in nutritional contents were observed in Nile tilapia cultured in a BF using diverse stocking densities 64 .www.nature.com/scientificreports/

Bacterial analysis
Heterotrophic bacterial counts were higher in the LD water than those detected in the HD water (p > 0.05; Table 6).The elevated bacterial counts in water samples at both densities were expected, as the BFT system enhances the multiplication of advantageous heterotrophic bacteria [65][66][67] .Similar results were reported by Arias-Moscoso et al. 68 , where heterotrophic bacterial counts in the shrimp-rearing BF system at day 30 ranged from 38.2 to 65.3 × 10 6 CFU/mL.In contrast, the Vibrio bacterial count in the water was significantly higher in the HD than in the LD (p = 0.02; Table 6).Arias-Moscoso et al. 68 also reported similar findings on day 30, ranging from 1.67 to 4.23 × 10 3 CFU/ mL, through the supplementation of commercial probiotics in a shrimp BF system.Enterobacter cloacae and Enterobacter amnigenus were identified in this study in the water of both HD and LD systems.Enterobacter spp. is a natural commensal of the animal gastrointestinal tract microorganisms 69 .Its presence in water samples in both studied system densities might be attributed to the accumulation of shrimp waste in the BF system.
Shrimp samples were analyzed to assess the effect of variable densities on shrimp food bacterial quality.The total bacterial counts in the HD system documented about a 1-log increase compared to the LD system.Similarly, the Vibrio-like bacterial count was markedly higher in shrimp samples from the HD compared to the LD (Table 6).The same patterns of Vibrio-like bacterial counts were found in the water from both systems, which might reflect on shrimp.Vibrio bacteria naturally exist in water environments and the gastrointestinal tract of shrimp 70 .Additionally, pathogenic Vibrio spp.causing food-borne infections were not identified in water or shrimp in both systems, while pathogenic Salmonella enterica subsp.arizona was identified in shrimp samples from the HD system (Table 6).

Gene expression
Generally, BFT contains a plentiful count of bacteria, and their cell wall consists of lipopolysaccharide, β-1, 3-glucans, and peptidoglycan.Consequently, it exhibits a probiotic effect and is recognized as a stimulant for the non-specific immunity of shrimps 17,54 .The expression levels of immune-related genes, namely propo, TGase II, sod, lyz, sp, pen 3, and the growth-related gene rap-2a in shrimps of the LD group were significantly higher than those of the HD group (Fig. 1).Conversely, the expression of the stress-related gene hsp70 showed a significant increase in the HD group (Fig. 1).
The proPO-system is activated upon the recognition of specific structures of probiotics or pathogens by pattern recognition proteins.It involves a serine proteinase pathway that ultimately cleaves and activates the proPO 71 .The respiratory burst of hemocytes aids in the clearance of shrimp infections and is linked to the release Table 6.Heterotrophic, Vibrio-like bacterial count, and other bacteria isolated from water (log CFU/mL) and white-leg shrimp (Litopenaeus vannamei) samples (log CFU/g) in biofloc systems with varying density.Data are expressed as means ± SE. www.nature.com/scientificreports/ of numerous reactive oxygen intermediates (ROIs) [72][73][74] .ROIs are considered essential for host defense, but their overexpression could potentially harm host cells 75 .SOD functions to maintain the lowest practical levels of ROIs intracellularly 76 .The documented impact of probiotics and BFT on enhancing the shrimp's respiratory burst has been reported 10,77,78 .
TGase is known to play a crucial role in the mechanism of blood coagulation, implicated in the defense mechanisms of invertebrates.TGase is broadly expressed in hematopoietic tissue and hemocytes, and the presence of lipopolysaccharide induces the rapid release of TGase as a response of hemocytes 79,80 .The upregulation of TGase in the LD group, along with the elevated lyz expression level, suggests that TGase is a significant component in the activation of the shrimp's immune response.Additionally, it is implicated in the modulation of antimicrobial peptides such as lysozyme 79 .
Other groups of antimicrobial peptides, including the penaeidins, are primarily expressed in hemocytes and provide defense against pathogens and infections 81,82 .Moreover, they contribute to opsonization by labeling bacterial surfaces, thereby enhancing immune reactivity and facilitating the elimination of marked antigens through phagocytosis 83 .Similarly, the expression levels of entire antimicrobial peptide gene groups were upregulated when bacterial probiotics were administered to shrimp, as documented by Antony et al. 84 .
In the higher-density group, hsp70 expression was significantly upregulated, indicating a correlation between shrimp stocking density and stress.This correlation is supported by Liu et al. 54 , who highlight the link between stocking density and stress.The evidence suggests that a Bacillus species mix can reduce cellular stresses in seabream larvae by decreasing the expression of hsp70, enhancing the fish's tolerance toward culture conditions 85 .Feed additives, immuno-stimulants, prebiotics, and probiotics are widely used in aquatic organisms to alleviate stress and enhance immune status 86,87 .

Digestive enzymes
Amylase, lipase, protease, and trypsin activities were estimated in the shrimp's hepatopancreases at the end of the experimental period (Fig. 2).The activities of enzymes (amylase, lipase, and protease) were significantly higher in the LD group than in the HD group, while no significant differences were observed in the activity of trypsin.www.nature.com/scientificreports/Various factors may influence the effectiveness of digestive enzymes in both fish and shrimp.Shao et al. 88 reported significant differences in trypsin activity in white-leg shrimp hepatopancreas samples fed different diets, with no notable differences in amylase and lipase activities.For instance, shrimp and fish fed with probiotics, microalgae, and periphyton-supplemented diets experienced increased levels of digestive enzyme activity 89,90 .An advantage of the BF system is its ability to enhance the digestibility capacity of reared shrimp by boosting the activity of digestive enzymes.The activities of digestive enzymes are critical factors for optimizing nutritional procedures and improving digestibility in aquatic animals, facilitating the breakdown and absorption of nutrients, growth enhancement, and adaptability to the environment 91,92 .This is consistent with the current results; the activities of the digestive enzymes amylase and lipase were lower in the HD (500 shrimp/m 3 ) group than in the LD (300 shrimp/m 3 ) group 54 .

Antioxidant enzymes
Antioxidant enzymes, including SOD and glutathione peroxidase, constitute the primary enzymatic defense against free radicals in organisms.When their levels decline, free radicals increase, leading to impairment of cell function 93 .In this study, antioxidant enzymes (catalase, SOD, glutathione reduced, and glutathione peroxidase) significantly increased in shrimp reared under the LD compared to the HD group (Fig. 3).
Conditions in the BF system positively contribute to the antioxidant activity in shrimp.Xu and Pan 5 observed that the antioxidant activity in shrimp subjected to two BF treatments was superior to that in the control group.Furthermore, Liu et al. 54 concluded that immunological response parameters and antioxidants, including SOD, glutathione peroxidase, and malondialdehyde, all decreased in higher density conditions (300 and 400 shrimp/ m 2 ).Emerenciano et al. 9 noted that BFT with a lower density system improved the antioxidant state of shrimp and reduced oxidative stresses, leading to better stress management during transfer into the hatchery and the reproduction process.The lower stocking density group exhibited lower oxidative stress levels compared to the higher stocking density group.These findings align with gene expression results, indicating a robust immunological and antioxidant response in low-density stocking.www.nature.com/scientificreports/

Histological assessment
Histological assessment (Fig. 4) revealed positive effects of the BF system on the health and structure of organs, as observed in the hepatopancreas, musculature, and gonad.Conversely, histological examination of gills indicated negative effects in the HD group, impacting the gill structure (Fig. 4).The hepatopancreas histology exhibited healthy hepatopancreas cells, lumen, nucleus, connective tissue, Blasenzellen cells (B), and Embryonal-Zellen cells.Musculature longitudinal and transverse sections displayed a normal structure.The female gonad showed a developed stage with peripheral cells, follicular cells, advanced vitellogenic oocyte, nucleus, and mature oocyte.In contrast, the gills in the lower density group maintained a normal structure with lamellae, uniform interlamellar space, and pillar cells.The higher density group, on the  The developmental status of the hepatopancreas can serve as a vital indicator of its functional activity 94 .The hepatopancreas performs essential functions such as lipid storage, nutrient absorption, and digestive enzyme production 95 .Blasenzellen (B) cells within the hepatopancreas are the primary and largest producers of digestive enzymes, responsible for nutrient accumulation, intracellular digestion, and transportation of digested material 96 .Shi et al. 97 demonstrated that the dietary microbial-derived antioxidant can enhance hepatopancreas functional activity through improved enzyme production.BF, recognized as a high-quality continuous food source, has been associated with improved production outcomes, shorter growth times, better survival rates, and well-structured musculature 98,99 .
The energy required for reproduction may be higher in females reared under BFT, and reduced glutathione levels are critical for organisms facing occasional oxidative stress 97,100 .Xu and Pan 41 highlighted the beneficial effects of BF as a potential dietary source of antioxidants for white-leg shrimp.It is evident that BFT enhances reproduction, aligning with the histological results of female gonad development in both treatments.
Shrimp gills play a crucial role in breathing, osmotic regulation, and ionic regulation 101 .Obstruction of gills by microorganisms as well as accumulation of inorganic nitrogen compounds can adversely affect shrimp breathing and osmoregulation by damaging the organ's structure 102,103 .Abnormal gill structure, such as malformation and clubbing lamellae, was evident in the higher-density group.In intensive aquaculture systems with no water exchange, shrimp mortality was linked to gill occlusion and high solid content in the water 103,104 .Total suspended solids and BF levels were found to positively correlate with stocking density, leading to an increased incidence of shrimp with stuffed gills 104 .Fregoso-López et al. 103 associated histological changes with more damage in shrimp gills at elevated stocking density, contributing to shrimp mortality.These findings may explain the significantly advanced survival rate in the lower-density group in the current study.

Conclusion
The impact of stocking density on shrimp farming using a BF system was significant.A comparison between two BF systems, one with a stocking density of 50 shrimp/m 2 and the other with 200 shrimp/m 2 , highlighted several drawbacks associated with higher stocking density.Growth performance parameters were reduced, the FCR increased, and the survival rate decreased.Additionally, higher counts of Vibrio-like bacteria in water and shrimp, along with the detection of Salmonella enterica ssp arizonae, were observed.A decline in body composition was also noted.Moreover, immune and growth-related genes showed lower expression, while stress genes exhibited increased expression.Reduced activity of digestive enzymes, antioxidants, and abnormal histological gill structure were additional findings.Further studies exploring varied inputs and operational methodologies for high-density BF systems could lead to highly productive production without undesirable consequences.

Figure 3 .
Figure 3. Activity of antioxidant enzymes (catalase, glutathione reduced, glutathione peroxidase, and superoxide dismutase) in the hemolymph of white-leg shrimp (Litopenaeus vannamei) reared under varying stocking densities using biofloc system.Data are expressed as means ± SE.

Table 1 .
Proximate analysis of the experimental feed and carbon sources (wheat flour).Values are presented as means ± SE.NFE = % DM-(CP + EE + CF + ASH).Data are expressed as means ± SE.

Table 2 .
Sequences of primers used in real-time PCR.

Table 4 .
Growth performance, survival rates, and feed utilization of white-leg shrimp (Litopenaeus vannamei) over a 16-week period, with variations in stocking densities within a biofloc system.Data are expressed as means ± SE.

Table 5 .
Proximate analysis of white-leg shrimp (Litopenaeus vannamei) and biofloc particles collected from biofloc systems with varying stocking densities.Data are expressed as means ± SE.