Overall evaluation of the replacement of fermented soybean to �sh meal in juvenile white shrimp, Litopenaeus vannamei diet: growth, health status, and hepatopancreas histomorphology

This study was conducted to determine the effect of replacing �shmeal (FM) with fermented soybean meal (FSBM) for 12 weeks on the growth performance, feed utilization, immunological parameters, antioxidant enzymes assays and lipid peroxidation, digestive enzymes, and histopathological analysis of juvenile Litopeneaus vannamei (L. vannamei). By substituting 0.0%, 20%, 30%, and 40% FSBM for �shmeal (w/w), four isonitrogenous diets were generated. A total of 300 juvenile L. vannamei(1.59 ±0.01 g) were randomly allocated to the experimental �ber tanks at a rate of �fteen shrimp per tank, with three replicates for each treatment. Growth performance and feed utilization decline considerably (P < 0.05) with increasing amounts of FM replacement with FSBM in diets. In comparison to the juveniles fed the other experimental diets, the diet containing a moderate level of FM replacement (20% FSBM) considerably enhanced growth performance and feed consumption during the feeding trial. The 20% FSBM-fed group had the highest protein content. In contrast, raising FSBM levels signi�cantly increased lipid content (P < 0.05) compared to the control. However, there were no statistically signi�cant differences (P> 0.05) across FSBM treatments. Hemolymph plasma total protein (TP) concentration and lysozyme activity were substantially greater (P < 0.05) in 20% FSBM compared to 40% FSBM (P < 0.05). In addition, 20% FSBM exhibits a substantial (P < 0.05) increase in the activity of antioxidant enzymes (CAT SOD, GPX, and GR). In contrast, the control and 30% FSBM groups had considerably more lipid peroxidation marker (MDA) than the 20% and 40% FSBM groups. Hepatopancreas amylase activity was considerably elevated (P < 0.05) in the control group and with 40% FSBM. In addition, Hepatopancreas and intestinal protease and lipase activity increased signi�cantly by 20% FSBM. Considerably more B-cells were present in the 40% FSBM diet than in the control diet, however they were signi�cantly less prevalent in the 20% and 30% FSBM of diets (P < 0.05).


Introduction
Crustaceans are a common species that contribute signi cantly to the aquaculture industry.Because of the high protein and minerals, it provides for healthy living, shrimp has emerged as the favorite and fastest-growing species in the crustacean aquaculture industry (Bondad-Reantaso et al., 2012).Paci c whiteleg shrimp (Litopeneaus vannamei; L. vannamei) is an important economic species in aquaculture (Pauly and Froese, 2012).In 2020, whiteleg shrimp was ranked as the top-produced species with 5. 81 million metric tonnes (FAO, 2022).The selection of shrimp species for culture is highly dependent on their ability to accept a wide range of feed formulations and ingredients (Chi et al., 2009).Despite its numerous advantages, the aquaculture industry faces numerous challenges, the most pressing of which is the demand for and supply of shmeal (FM), the industry's primary protein source in arti cial aquatic and marine shrimp diets.FM has long been used in the aquafeed formulation as a source of high-quality protein with its high digestibility and good amino acid (AA) pro le (Sookying et al., 2013).However, reliance on FM has been identi ed as a signi cant impediment to the aquaculture industry's long-term development (Tacon and Metian, 2008) because of its consistent price increase due to its limited supply and increased incorporation in livestock and aquaculture feed (Zhang et al., 2018).As a result, pursuing less expensive and more sustainable alternative protein sources of both animal and plant origin to reduce FM levels in aquafeed without impairing growth performance is a major ongoing global interest (Ding et al., 2015;Faggio et al., 2015;Dossou et al., 2018;Hoseinifar et al., 2018;Iswarya et al., 2018;Nath et al., 2018 andRingo et al., 2018).Over the last few decades, a wide range of protein ingredients has been studied as potential FM replacers, with plant proteins receiving the most attention as the most viable candidates due to their lower cost and abundant availability (Gatlin et al., 2007 andDossou et al., 2018 and Abd El-Naby et al., 2022).Plant protein sources such as soybean meal (SBM) are the primary options when replacing FM in aqua-feed.However, SBM has several nutritional drawbacks, including the presence of anti-nutritional factors (ANFs) that reduce feed utilization, absorption, and feed conversion ratio (Kikuchi, 1999).In addition to the amino acid imbalance and lower protein content when compared to FM. Thermal and mechanical processes, soaking, germination/malting, and fermentation have all been used to reduce the ANFs content of SM and improve its nutrients, bioavailability, and nutritional value (Hotz and Gibson, 2007).Among these methods, fermentation has been proposed as the most costeffective for improving the nutritional quality of SBM not only through the biodegradation of ANFs (such as trypsin inhibitors, oligosaccharides, and phytic acid), proteins, and bers but also through the production of probiotics and prebiotics, which may improve palatability, nutrient digestibility, and immune function (Hong et al., 2004).Fermented soybean meal (FSBM) was produced by fermenting SBM; many microorganisms were tested for SBM fermentation, and it was revealed that the nutritional value of the produced FSBM varies depending on the type of microorganism (Feizi et al., 2022).Thus, the goal of this study is to assess the effects of long-term feeding fermented soybean meal in different levels of replacement to the sh meal on juvenile L. vannamei.Growth performance, feed utilization, immunological parameters, antioxidant enzyme assays, lipid peroxidation, digestive enzymes, and histopathological analysis were measured for providing accurate evaluation.

Ethical statement
The Animal Research and Ethics Committee of the National Institute of Oceanography and Fisheries in Suez, Egypt approved the research work plan and permitted it to work with Litopenaeus vannamei postlarvae.All experiments were done by the committee's rules.

Solid-state fermentation of soybean meal preparation
Commercial soybean meal (SBM) was obtained from a company in Egypt's Zagazig province and ground to a particle size of 500 m.The modi ed method of Yabaya et al (2009) was used to perform fermented SBM.Brie y, two kilograms of SBM were combined with 1.1 liters of distilled water (50% moisture), and 60.5 mg of commercial dry yeast Saccharomyces cerevisiae (S. cerevisiae) of cell density 3 ×10 6 cell g 1 (Fermipan®, GB ingredients, China).In a Hobart food mixer, all the ingredients were homogenized for 15 minutes.The mixture was incubated for 48 h at 40 °C, the ideal growth temperature for S. cerevisiae, in a 10 L glass jar covered with aluminum foil.Ten grams of the solid-state fermented soybean meal (SSF-SBM) with yeast was sampled at 0, 12, 24, and 48 h of fermentation to assess the anti-nutritional factors (ANFs) and chemical composition content.Finally, the SSF-SBM was dried to a constant weight at 70 °C.

Diet formulation
A control diet with FM (FSBM0) as the primary source of protein and three experimental diets in which the FM in the control diet was replaced by FSBM at 20, 30, and 40% resulted in four isonitrogenous (41.2 g/kg crude protein and 8.2 g/kg isocaloric diets) (Table 1).The dry ingredients and sh oil were combined to make a stiff dough by adding water.Using a meat grinder, the mixtures were formed into 1.6 mmdiameter pellets.In a forced-air oven set at 40°C, pellets were dried.Before use, fully dried diets were put in plastic bags and kept at 18 °C.

Amino acid analysis
Following procedures recommended by the Association of O cial Analytical Chemists (AOAC, 1990) and the protocol of Llames and Fontaine (1994), the amino acid pro le of FSBM was determined using an amino acid analyzer (LA8080, Amino SAYA, Hitach High tech, Japan).

Feeding trial
L. vannamei juveniles were obtained from the El-Sahaba hatchery in Damietta, Egypt, and transported to the National Institute of Oceanography and Fisheries' invertebrates Laboratory in Suez, Egypt.The juveniles (1.59 ± 0.01 g) were acclimated for one week before being distributed at random into fteen berglass tanks (40 L volume).Compressed air was used to provide continuous aeration.For 12 weeks, shrimp juveniles were fed the four test diets in triplicate (0.0, 20%, 30%, and 40% FSBM, respectively) to apparent satiation twice daily.Dietary amounts were adjusted based on survival and body weight.
Approximately 10% of the water volume in each aquarium was changed out daily with fresh aerated and ltered marine water after removing the accumulated wastes.

Physicochemical characteristics of water
Daily partial exchange of 10% of the water kept the water quality parameters within the acceptable ranges during the experimental trial.Dissolved oxygen levels were kept at 5.74 ± 0.50 mg/L using an oxygen meter (HANNA, HI 9146-04/10), pH was kept at 7.5 ± 0.3 using a pH meter (Adwa, AD 11), unionized ammonia concentration was kept at 0.22 ± 0.04 mg/L using DREL/2 HACH kits (HACH Co., Loveland, Co.), and salinity was kept at 30.56 ± 0.80 (Bioevopak Co., Ltd, China).While the water temperature was maintained at a range of 28.52 ± 0.92 °C.

Growth performance and feed utilization:
Weight in (g) and length in (cm) of each post larvae (PL) were measured at the start of the experiment and every two weeks for the next 12 weeks.All shrimp were weighed to determine their nal weight at the end of the experiment, which was then compared to their initial weight on the rst day.By counting the individuals in each aquarium, the survival rates of the PL were also estimated.

Feed and body composition chemical analysis
The standard methods of AOAC, (2012) were used to determine the moisture, crude protein, crude lipid, and ash content of diets and shrimp samples.The dry matter of the samples was determined by drying them to a constant weight at 105 °C.Crude protein was calculated by multiplying nitrogen by 6.25 using the Kjeldahl method (Kjeltec TM8400, FOSS, Sweden).The Soxhlet method was used to determine crude lipid after diethyl ether extraction (Buchi 36680, Switzerland).After 16 hours of combustion in a mu e furnace at 550°C.According to Goering and Van Soest (1970), the crude ber was estimated.Gross energy was computed according to the standards of NRC (1993).

Immunological parameters
Hemolymph samples were taken from the ventral sinus of the recently molted shrimp using 1-ml syringes, and a 27-gauge needle containing anticoagulant (1:1), according to El Asely et al. (2010).The molting stages were identi ed through periodic observation of the ock and were con rmed through microscopic examination of the epidermal retraction following Robertson et al. (1987).
Hemolymph plasma was obtained by centrifugation to the hemolymph-anticoagulant mix at (10000 g/4 o C) for 5 minutes, according to Lamela et al. (2005).The obtained plasma was stored at -20 o C.
The total protein of hemolymph plasma was determined following Bradford, (1976) at 595 nm, where the bovine serum albumin was used as standard.
Lysozyme activity was determined through measurement of the decrease in the absorbance of lysis to the Micrococcus lysodeikticus cells (Sigma-Aldrich, Cat.no.LY0100) at a wavelength of 450 nm following the manufacturer instructions and the protocol of Sotelo-Mundo et al. (2003).

Antioxidant enzymes assays and lipid peroxidation
The shrimp were euthanized following the American Veterinary Medical Association (AVMA) Guidelines for Animal Euthanasia (Leary et al., 2013).Shrimp were immersed in ice for 15 minutes till no motion was noticed.Hepatopancreas was dissected and kept in cold phosphate buffer saline (PBS), and then it was homogenized.Tissue homogenate was centrifuged at 12,000 ×g for 12 min at 4 °C.The supernatant was removed and stored at −75 °C.Catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (Gpx) activities were measured photometrically using a microplate reader and the commercially available kits (Abcam; Catalase-ab83464, SOD-ab65354, GPx-ab219926) at the wavelength (OD 570 nm, OD 450, and OD 405 nm) respectively.While GR activity was measured uorometrically at Ex/Em = OD420/480 nm using (Abcam; GR-ab83461) kits.Colorimetric lipid peroxidation Assay Kit (Abcam, ab118970) was used to detect the content of Malondialdehyde (MDA) in the hepatopancreas tissue homogenate at OD = 532 nm.

Digestive enzymes assay
Hepatopancreas and intestines were weighed and homogenized in cold PBS.Tissue homogenates were centrifuged at 18894 at 4°C for 5 min.The supernatant was stored at −20 °C until analysis.Amylase, lipase, and protease activity were determined using Abcam kits following the manufacturer's instructions.Amylase activity was detected using (ab102523) colorimetric kits measured at OD = 405 nm.Lipase activity was assayed using (Abcam, ab102524) kits, where lipase hydrolyzes a triglyceride substrate to form glycerol which is quanti ed enzymatically by monitoring a linked change in the absorbance of a probe (OD=570nm).Lipase activity was calculated as nmol of glycerol per mg protein.Protease activity was estimated by Protease Activity Assay Kit (Fluorometric -Green) (ab112152) with uorescence intensity at Ex/Em = 490/525 nm.

Histopathological analysis
The hepatopancreas of (three shrimp/replicate/group) were dissected, then xed for 24 hours in Davidson's xative before being transferred to 70% ethanol for standard histological processing (Bell and Lightner, 1988).The tissue sections (4-5m) were stained with hematoxylin-eosin, examined using a light microscope (Olympus CX 41, Japan), and photographed using an Olympus E-620 digital camera.Using (Image J software) equipped with (a cell counter plugin), the number of R-cells (Restzellen or Resorptive cell) and B-cells (Blasenzellen or Blister cell) as well as tubule diameter were determined in 20 randomly selected tubules from each treatment (Romano et al., 2015).
2.12.Statistical analysis SPSS software (Ver17.0)was used to conduct the statistical analysis.All data were given as mean ± standard error (S.E.).Using one-way ANOVA and turkey's post hoc multiple comparisons, statistical signi cance was determined.P-values < 0.05 were deemed statistically signi cant.

Essential amino acid content
In the current study, the partial replacement of FM with FSBM in uenced the aa pro le in the diet, with an improvement in the contents of some amino acids such as arginine, lysine, leucine, isoleucine, phenylalanine, threonine, and valine in shrimp diets, and a decrease in the contents of some amino acids such as histidine and methionine (Table , 2) 3.2.Growth performance and feed utilization At the end of the feeding trial, the survival of L. vannamei juvenile was reduced with no signi cant difference between feeding groups.There was no signi cant difference in growth performance or feed utilization parameters between the four experimental groups (P > 0.05).(Table 3).The 20% FSBM diet had signi cantly improved growth performance in terms of FBW, BWG, RBWG, and SGR (P < 0.05), followed by the groups fed the (30, 0.0, and 40% FSBM diets, respectively).While there were no statistically signi cant differences in these parameters between the 30, 0.0, and 40% FSBM diets.The lowest was found at 40% FSBM.
As shown in table (4), the low replacement level (20% FSBM) signi cantly increased feed intake, resulting in the greatest growth performance relative to the other replacements.In addition, 20% replacement exhibited the lowest FCR (P <0.05) compared to those fed a 40% FSBM diet.Conversely, the low replacement level group (20% FSBM) signi cantly improved (P <0.05) FER, PER, APU, and EU compared to the high replacement level group.40% FSBM diet produced the least amount of feed utilization,

Proximate body composition analysis
Table (5) presents the approximate composition of the entire body of L. vannamei juveniles.The present investigation revealed no statistically signi cant variations (P > 0.05) in the moisture content of L. vannamei shrimp across all dietary regimens, with moisture levels ranging from 75.64 to 76.46%.The group fed 20% FSBM had the highest protein content (21.99%), while the group fed 0% FSBM had the lowest protein level (20.36%).It was noticed that raising FSBM levels signi cantly increased lipid content (P < 0.05) compared to the control.However, there were no statistically signi cant changes (P > 0.05) amongst FSBM treatments.In contrast, as the amount of dietary FSBM increased from 0 to 40% FSBM, ash concentrations tended to decrease.The highest signi cant levels of ash content were recorded in the control diet (0.0% FSBM).

Antioxidant enzymes assays and lipid peroxidation
The obtained results presented in table (7) showed a signi cant increase (P < 0.05) in the activity of hepatopancreas catalase enzyme in 20% FSBM, the same pattern was recorded in the other measured antioxidant enzymes (SOD, GPX, and GR).On the other hand, signi cantly low catalase activity was recorded in 30% FSBM, followed by control and 40% FSBM.In contrast, the content of lipid peroxidation marker (MDA) was signi cantly higher (P < 0.05) in control and 30% FSBM than in 20 and 40% FSBM levels.

Digestive enzymes assay
Hepatopancreas amylase activity was signi cantly high (P < 0.05) in 40% FSBM and control followed by 30% FSBM and 20% FSBM (Table, 8).While, protease activity showed a signi cant increase in 20% FSBM, and its lowest activity was recorded in 40% FSBM.While protease activity showed a signi cant increase in the 20% FSBM diet, and its lowest activity was recorded in the 40% FSBM diet.
Contrary, the values of Lipase activity were signi cantly higher (P < 0.05) in 20% of FSBM, and the lowest activity was recorded in 40% of FSBM (table 8).
On the other hand, intestinal amylase activity was signi cantly high (P < 0.05) in 0.0% FSBM while, 20% FSBM was the lowest (Table, 8).Protease activity showed a signi cant increase in 20% FSBM, and its lowest activity was recorded at 40% FSBM.Contrary, the values of Lipase activity were signi cantly higher (P < 0.05) in 20% of FSBM, and the lowest activity was recorded in 40% of FSBM (table 8).

Gross pathology
Gross pathology revealed that shrimp given 30 and 40% FSBM exhibited a pale white distal hepatopancreas and a slightly shrunken stomach (Fig 1a and b).

Histology of Hepatopancreas
Figure 2 depicts the hepatopancreatic tubules of shrimp fed the control and experimental diets (a-d).Considerably more B-cells were present in the 40% FSBM diet than in the control diet, however, they were signi cantly less prevalent in the 20% and 30% FSBM diets (P < 0.05).In addition, 30% of FSBM-fed shrimp had considerably more R-cells than the others.In terms of hepatopancreatic tubule diameters, the 30% and 40% FSBM diets had considerably larger tubules than the other diets (P < 0.05).The predominance of B-cells and R-cells in tubules, as well as the tubule diameter, are shown in Table 9.

Discussion
Plant proteins specially soya bean (SB) could be a promising source of protein in Aquatic animal feed, however, they are high in cellulose, which is di cult for sh and other monogastric animals to digest (Gatlin et al., 2007).Researchers are working on bioprocessing SB in other products such as soybean meal (SBM), soy protein isolate (SPI), fermented soybean meal (FSBM), and soybean protein concentrate (SPC) in shrimp diets in order to reduce ANFs (Abdul Kader  The ndings of this study indicate that 20% of FM can be substituted with FSBM without harming the health of shrimp.In addition, juvenile L. vannamei given 20% FBSM displayed enhanced growth performance and feed utilization, despite the fact that the amino acid composition of all experimental groups was identical.In addition, this improvement may be attributable to the improved lipid digestibility of FSBM (Refstie et al., 2005) and the reduction of anti-nutritional components during fermentation (Su et al., 2018).In contrast, the growth of shrimp fed a diet containing 40% FSBM was diminished.This decline may be attributable to the presence of non-digestible oligosaccharides, decreased protein digestibility, or a nutritional imbalance (Sharawy et al., 2016).
Our study's growth performance results were comparable to those of earlier research conducted on other crustacean species.According to the ndings of Ding et al. (2015), the optimal growth performance of M. nipponense was achieved when 25% of the FM was substituted with FSM.Research on F. indicus suggests that replacing up to 28.57 percent of FM with FSM is signi cantly more economical (Sharawy et al., 2016).Shao et al. (2018) observed that a meal with a moderate FSM replacement level (20% of FM protein) e ciently boosted the growth of juvenile white shrimp and that a replacement level of up to 40% had no in uence on shrimp growth performance.
In the present investigation, there were no statistically signi cant differences (P>0.05) in the moisture content of L. vannamei shrimp between diets.While the addition of S. cerevisiae increased the protein content of FSBM-fed groups signi cantly.The same rise in body lipid content was observed with increased dietary FSBM, which included more carbohydrates than FM (Makkar et al., 2007).According to Kaushik et al. (2004), one of the major factors leading to increased lipid retention is an increase in dietary plant protein, which is associated with an increase in hepatic lipogenic enzyme activity, imbalances in dietary amino acid content, and higher whole-body lipid levels in sea bass and salmonids.In addition, Sharawy et al. (2016) reported that there were statistically signi cant variations (P< 0.05) in the protein, dry matter, lipid, and ash content of shrimp fed different experimental diets compared to control diets containing 0.0% protein (FSBM).In our study, ash contents tended to decrease as FSBM levels increased from 0 to 40% of the meal.
Hemolymph metabolites serve as physiological, nutritional, and immunological stress indicators in crustaceans.In addition, it has been used to evaluate the nutritional health of shrimp, whose blood protein and glucose levels are very sensitive to the protein content of their diet (Rosas et al. 2001).In the current study, the inclusion of FSBM in the diet had a signi cant effect on the hemolymph total protein (TP) content, with the maximum TP concentration seen in groups fed 20% FSBM, followed by 30% FSBM.According to Shiu et al., (2015) the increased protein content of soya bean meal after fermentation may account for the observed results.On the other hand, the decreased TP concentration in 40% FSBM is due to the detrimental effect of high soya bean levels on the digestibility, absorption, and utilization of dietary protein (Gilani et al., 2012).
With its antibacterial effectiveness against bacterial infection, lysozyme activity is one of the most important indicators of shrimp immunity (Kaizu et al., 2011).The addition of S. cerevisiae to ferment SBM was also bene cial in regulating serum lysozyme activity.The results demonstrated a considerable increase in the lysozyme activity of 20% of FSBM-fed groups.The process involved in boosting the immune system of shrimp hinges on the protein recognition pattern of the circulating sugars which evoke the immune cells (Vargas-Albores and Yepiz-Plascencia 2000).It is assumed that the yeast harboring βglucan effectively stimulated lysozyme synthesis.In contrast to what was expected, the lysozyme activity decreased as the concentration of FM-replacement FSBM increased.This was postulated as a result of the fatigue of lysozyme-producing cells from long-term exposure to the triggering agents, recommending the use of a low dose for a brief period of time for an effective response (El-Barbary et al.In addition to exogenous sources of reactive oxygen species (ROS), regular cellular metabolism generates electrons that can alter the membrane protein structure, lipids, cell division, and apoptosis signaling pathway (Redza-Dutordoir and Averill-Bates, 2016; Bauer and Bauer, 1999).Antioxidants' function in cells is to maintain balance and scavenge excess reactive oxygen species (ROS) to mitigate their corrosive effect (Kurutas, 2015).
In the present study, hepatopancreas CAT, SOD, Gpx, and GR activities increased signi cantly in the group fed 20%FSBM, indicating that the 20% substituted FSBM meal had a greater anti-oxidative effect than sh-fed FM and the other two concentrations.Ding et al. (2015) detected a drop in CAT, SOD, and GSH-PX activities with increased FSM content in the diet of Macrobrachium nipponense.The results obtained were almost identical to those reported by Ding et al. which suggested that the anti-oxidative capacity of shrimp was compromised by the substitution of shmeal.Xu et al. (2008) found that sh CAT activity fell dramatically from 30% to 20% when shmeal was substituted.Despite the fact that Daiyong et al. (2009) discovered that the CAT activity of shrimp was unaffected, the SOD activity declined dramatically when shmeal was reduced from 25% to 20%.In addition to the enhanced avonoid content created during soybean fermentation, tiny peptides, organic acids, and probiotics are also produced (Mukherjee et al., 2016).Saccharomyces cerevisiae produces vitamins and other metabolites that serve as exogenous antioxidant sources (Farid et al., 2019).
The hepatopancreatic MDA level of shrimp given 20% FSBM was much lower than that of shrimp fed the FM and other diets, demonstrating that the dietary replacement of FM with FSBM did not stimulate oxidative stress and was even successful at decreasing it.This could be attributable to the FSBM's high iso avonoid content, which can neutralize free radicals and prevent lipid peroxidation (Yoon and Park, 2014).
The hepatopancreas is responsible for the generation and release of digestive enzymes, the absorption of nutrients, and the mobilization and transport of nutrients such as lipids, glycogen, minerals, and organic compounds to muscle and other tissues in response to growth and reproductive needs (Ceccaldi, 1989).The hepatopancreas secretes enormous quantities of digestive enzymes, such as amylases and proteases (Gamboa-delgado et al., 2003).Dietary content has a signi cant in uence on digestive enzyme production and activity (Le Moullac et al., 1997;Guzman et al., 2001).In the present investigation, the substitution of sh meal with FSBM had a substantial effect on the activity of digestive enzymes; amylase activity was signi cantly higher in 40% FSBM and control diets than in other diets, indicating a higher carbohydrate content.
Interestingly, the digestive enzyme concentration in the intestine was substantially identical to that of the hepatopancreas, corroborating the ndings of Córdova-Murueta et al (2003).
The hepatopancreas is the most essential digestive organ in shrimp.Histologically, it consists of four distinct cell types contained within blind-ending tubules.E-cells differentiate into R-cells (nutrient absorption and storage), F-cells (production of digestive enzymes), and B-cells (presumed to be secretory in function) at the apex of the tubules (Gopinath and Paul Raj, 2009).
In the present study, shrimp fed 30% partial sh meal replacement had signi cantly more R-cells than other groups (P < 0.05); this increase in R-cells, which are responsible for lipid storage in the hepatopancreas gland, may be indicative of an increase in energy reserve in the hepatopancreas as a result of the treatment.The B-cells are large cells responsible for enzyme storage, and this study revealed that their prevalence was signi cantly higher in the 40% partial sh meal replacement group than in the control group, but signi cantly lower in the 20% and 30% partial sh meal replacement groups (P < 0.05).In previous research, hypertrophied B-cells were identi ed in shrimp fed moderate dosages of the mycotoxin deoxynivalenol (DON), indicating oxidative stress in shrimp (Xie et al., 2018).Also, the increased B-cell prevalence in L. vannamei has been observed to increase at low salinities, suggesting that this is a response to the increased nutrient use required for higher osmoregulatory functions (Li et al., 2008).Similar to our ndings, Romano et al (2015) concluded that while the prevalence of R-cells was signi cantly higher in shrimp-fed organic acids-blended diets, indicating greater energy reserves, the prevalence of B-cells, which are primarily responsible for the secretion of digestive enzymes, was signi cantly lower.The 30% and 40% partial sh meal replacement groups had substantially larger hepatopancreatic tubule diameters than the other groups (P < 0.05).The increase in hepatopancreatic tubule diameter may be correlated with the greater prevalence of R-cells and the resulting fat storage inside them (Johnston et al., 2003;Simon and James, 2007;Pourmozaffar et al., 2019).Which may be associated with the gross pathology picture of the hepatopancreas with the white fuzzy zone.

Conclusion
In conclusion, the current study demonstrates that 20% replacement of FM with FSBM improved growth performance, feed utilization, immunological parameters, antioxidant enzymes assays, lipid peroxidation, digestive enzymes, and histopathological analysis in juvenile L. vannamei, despite the fact that there was no difference in the amino acid content between the experimental groups.This improvement may also be attributable to the increased lipid digestibility of FSBM and the decrease in anti-nutritional components during fermentation.

Declarations
. Tacon, A.G.J. and Metian, M. (2008).Global overview on the use of sh meal and sh oil in industrially compounded aquafeeds: Trends and future prospects.Aquaculture, 285(  Survival rate (%) = 100 × ( sh no. at the end/ sh no.stocked at the beginning); Table 4. Feed utilization of Litopenaeus vannamei post-larvae fed diets with different replacement ratios of shmeal (FM) with fermented soybean meal (FSBM) for 12 weeks.Note.
Means having the same letter in the same row is not signi cantly different at P<0.05 Feed intake (FI) = total feed intake per tank/number of sh; Feed conversion ratio (FCR) = feed intake (g)/body weight gain (g); Protein e ciency ratio (PER) = weight gain (g)/total protein intake (g); Apparent protein utilization (APU %) = 100 (protein gain in sh (g)/protein intake in diet (g)); Energy utilization (EU %) = 100 (gross energy gain (g)/ gross energy intake (g);   Means having the same letter in the same row is not signi cantly different at P<0.05.Table 9. B-cell and R-cell prevalence (number/tubule) and tubule diameter (μm) (Mean ± SE) from the hepatopancreas of shrimp Litopenaeu svannamei post-larvae fed diets with different replacement ratios of shmeal (FM) with fermented soybean meal (FSBM) for 12 weeks.

FSBM
. FM: sh meal; FSBM: fermented soybean meal.Means having the same letter in the same row is not signi cantly different at P<0.05

Figures Figure 1 a 2
Figures

Table 3 .
Growth performance of Litopenaeus vannamei post-larvae fed diets with different replacement ratios of shmeal (FM) with fermented soybean meal (FSBM) for 12 weeks.

Table 5 .
Whole body composition (%, of fresh weight basis) of Litopenaeus vannamei post-larvae fed diets with different replacement ratios of shmeal (FM) with fermented soybean meal (FSBM) for 12Means having the same letter in the same row is not signi cantly different at P<0.05.