Effects of Stocking Density and FeedingfFrequency on the Growth Performance, Digestive Enzyme Activity, and Tissues Histology of Hybrid Grouper (Epinephelus Fuscoguttatus♀×E. Lanceolatus♂)

Effects of Stocking Density and FeedingfFrequency on the Growth Performance, Digestive Enzyme Activity, and Tissues Histology of Hybrid Grouper (Epinephelus Fuscoguttatus♀×E. Lanceolatus♂) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research article Effects of Stocking Density and FeedingfFrequency on the Growth Performance, Digestive Enzyme Activity, and Tissues Histology of Hybrid Grouper (Epinephelus Fuscoguttatus♀×E. Lanceolatus♂) Yan Chen, Hai Huang, Jun Ma, Wenkan Liu, Honggan Zhong, Jiechao Zhang, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-103762/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract A 6-week 5×2 factorial study was conducted to examine the effects of stocking density and feeding frequency on growth performance, body composition, digestive enzyme activity, and tissue histology of juvenile hybrid groupers raised in an indoor circulating water system. Triplicate groups of fish were reared in tanks following a factorial design consisting of ten treatments including 5 stocking densities (SD) [1.1 kg m −3 (0.55 kg fish per tank, SD1.1), 2.2 kg m -3 (1.1 kg fish per tank, SD2.2), 3.3 kg m −3 (1.65 kg fish per tank, SD3.3), 4.4 kg m −3 (2.2 kg fish per tank, SD4.4) and 5.5 kg m −3 (2.75 kg fish per tank, SD 5.5)] and 2 feeding frequencies (FF) [3 meals a day (FF3) and 2 meals a day (FF2)]. The resulting 10 treatments were G1(SD1.1,FF3), G2(SD2.2,FF3), G3(SD3.3,FF3), G4(SD4.4,FF3), G5(SD5.5,FF3), G6(SD1.1,FF2), G7(SD2.2, FF2), G8(SD3.3, FF2), G9(SD4.4, FF2) andG10 (SD5.5, FF2). Feed consumption and temperature were recorded throughout the experiment. After 6 weeks, the results indicated that the weight gain rate (WGR) and specific growth rate (SGR) of fish in the G7 group were significantly higher than those of other groups ( P < 0.05), followed by G1, with G3 being the lowest. Weight gain and specific growth rates were generally higher in fish fed twice a day than those fed three times a day. The variations in protein content between groups were consistent with the muscle protein content trends. Feeding frequency and stocking density had significant effects on serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), triglycerides (TGs), and cholesterol (CHO) ( P < 0.05). Regarding immune function, grouper albumin (ALB), serum lysozyme (LZY), superoxide dismutase (SOD), and alkaline phosphatase (AKP) were significantly affected by stocking density and feeding frequency ( P < 0.05). Pepsin and lipase activities in the stomach, intestine, and liver were also affected. The histological structure of the stomach, liver, and intestine in G1, G2, G7, and G8 fish was relatively normal, whereas those of the remaining groups exhibited varying degrees of damage. Overall, the optimum stocking densities were 1.343 kg/m 3 (approximately 10 fish) and 2.004 kg/m 3 (approximately 20-30 fish) when the fish were fed 3 and 2 times per day, respectively. Medical Genetics Molecular Genetics Population Genetics hybrid grouper stocking density feeding frequency growth performance digestive enzyme activity tissue histology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background The balance between rapid fish growth, feed utilization efficiency, feeding strategy, and intensive fish-farming practices is a key point in fish aquaculture [ 1 , 2 ]. Feeding time and frequency have been reported to affect feed intake and growth performance in different fish species including the Australian snapper Pagrus auratus [ 3 ], pikeperch Sander lucioperca [ 4 ], African catfish Clariasgariepinus, Burchell 1822 [ 5 ], flounder Platichthysflesusluscus [ 6 ] and rock bream Oplegnathusfasciatus [ 7 ]. Water quality, management procedures, stocking density, feeding frequency, and nutritional conditions are all directly associated with fish stress [ 8 , 9 , 10 ]. Feeding frequency is a well-known regulator of growth performance, feed intake, and chemical composition of fish, and therefore optimizing this factor can substantially reduce aquaculture production costs and prevent water quality deterioration resulting from excess feeding. In practice, fish are fed by most farmers to satiety based on visual observation. However, it is very difficult to identify the point at which fish are satiated and fish are overfed frequently. The stomach and intestine may exceed their capacity as a result of overfeeding, which leads to decreases in digestive efficiency and feed utilization [ 11 ]. On the other hand, an insufficient feeding frequency leads to poor growth and high mortality, particularly in intensive systems[ 12 ]. For example, sporadic and low feeding rates may lead to slower growth rates, increased hunger, intraspecies aggression, and increased cannibalism [ 13 ]. Intensive fish farming practices often result in fish stress and poor health. In contrast, improved feeding conditions not only benefit the welfare of fish but also aquaculture profits [ 9 ]. In most fish species, high stocking densities adversely affect fish survival, weight gain, and specific growth rates[ 14 ]. These effects are attributed to the competition for food and/or space resulting from food scarcity [ 15 ], which leads to the establishment of hierarchy [ 16 ]. Additionally, high stocking densities can increase the likelihood of physical injuries, stress, and susceptibility, and can also modulate swimming and aggressive behaviors[ 17 ]. There is considerable evidence of welfare decline in rainbow trout Oncorhynchus mykiss stocked at high densities; however, excessive aggression and poor feeding rates may also occur when rainbow trout are reared at very low densities [ 18 ]. Hybrid grouper ( Epinephelus fuscoguttatus♀×E. lanceolatus♂ ) is among the most widely farmed fish species worldwide, particularly in China. Although some work has been done regarding the effects of temperature, nutrients, and disease on grouper growth [ 19 , 20 , 21 , 22 , 23 ], studies on the effect of feeding frequency and stocking density on this species in Chinese waters remains scarce. Moreover, the synergistic effects of both parameters have not been characterized. Therefore, our study sought to evaluate the effects of stocking density and feeding frequency on growth performance, body composition, digestive enzyme activity, and histology of juvenile hybrid groupers, with the overarching goal of determining whether these two stressful fish farming practices affect fish physiology synergistically or individually. 2 Methods 2.1 Experimental diets Diet formulation and proximate analysis [Association of Official Analytical Chemists (AOAC), 1984][ 24 ] details are provided in Table 1. The diet ingredients including fishmeal, soy protein concentrate, casein, shrimp meal, wheat flour, binding agents, soybean oil, fish oil, squid visceral ointment, choline chloride, and monocalcium phosphate were acquired from Guangzhou Rifeng Science & Technology Co., LTD. The mineral and vitamin premixes were purchased from Guangzhou Ashare Aquatech Co., Ltd All dry ingredients were first thoroughly mixed in a blender, after which the oil was slowly added to the mixture while blending. Deionized water (300 mL kg − 1 dry mixture) was then slowly mixed in to achieve a dough-like consistency. This dough was then extruded into pellets (3 mm in diameter) using a twin-screw extruder (South China University of Technology F-26 (II)) and dried in a 60 ℃ oven until the moisture content was less than 8%. All pellets were sealed in plastic bags and frozen (− 20 ° C) until used. 2.2 Experimental fish and feeding trial Prior to the experiment, the use of animals was approved by the "Institutional Animal Care and Use Committee of Hainan Tropical Ocean University" and "Hainan Key Laboratory for Conservation and Utilization of Tropical Marine Fishery Resources" (20161111A1). Juvenile hybrid groupers were acquired from a fish farm (Hainan Chenhai Products Co., Ltd.). The fish were originally kept in a high-quality well water circulation system, after which they were randomly assigned to 30 outdoor circular tanks (500 Litres) that were continuously supplied with 30 liters of water per minute. The following were the inflow water quality parameters: 27.0–28.9 ℃; 7.0–8.2 mg L − 1 of dissolved oxygen; pH 6.8–7.5; 28.1–31.1‰ salinity; and 0.05–0.1 mg/L total ammonia nitrogen. Feed intake and fish mortality were recorded daily. Two variables were considered together: feeding frequencies (FF) [3 meals a day (FF3) and 2 meals a day (FF2)] and stocking density (SD) [1.1 kg m − 3 (0.55 kg fish per tanks, SD1.1), 2.2 kg m − 3 (1.1 kg fish per tank, SD2.2), 3.3 kg m − 3 (1.65 kg fish per tank, SD3.3), 4.4 kg m − 3 (2.2 kg fish per tank, SD4.4), and 5.5 kg m − 3 (2.75 kg fish per tank, CD5.5)]. Based on this 5 × 2 factorial design, ten treatments were analyzed in triplicate randomly: G1(SD1.1,FF3), G2(SD2.2,FF3), G3(SD3.3,FF3), G4(SD4.4,FF3), G5(SD5.5,FF3), G6(SD1.1,FF2), G7(SD2.2,FF2), G8(SD3.3,FF2), G9(SD4.4,FF2), and G10(SD5.5,FF2). The trial was conducted for 42 days without counting a 15-day pre-trial acclimation period. 2.3 Sample collection At the end of the growth test, the fish were collected after 24 hours of fasting, anesthetized with tricaine methanesulfonate (MS222) (50 mg L − 1 ), and weighed/measured. Fish numbers and weights per tank were recorded to calculate the feed efficiency ratio (FER), specific growth rate (SGR), survival, and protein efficiency ratio (PER). Eight fish were randomly selected from each tank for proximate analysis, three for whole-body composition tests and five for white muscle components. The viscera, liver, and intraperitoneal fat of fish were quickly removed and weighed to calculate hepatosomatic index (HSI), viscerosomatic index (VSI), and intraperitoneal ratio (IPR). Blood samples were taken from the tail vein of eight fish in each tank with syringes and centrifuged at 3000 g for 10 minutes at 4 ℃. 2.4 Evaluation of growth performance The main growth performance variables were calculated as follows: WG (%) = 100 × (final mean weight - initial mean weight)/initial mean weight (g) Survival (%) = 100 × final fish number/initial fish number Average body weight gain (BWG, g) = (W f − W i )/number of fishes; Specific growth ratio (SGR, % d−1 ) = 100 × (In Wf − Wi)/t; Weight gain ratio (WGR) = (W f − W i )/W i ; Daily feed intake (DFI, % d − 1 ) = 100 × dry feed intake/[(initial feed weight + final weight)/2 × t]; Condition factor (CF, g cm − 3 ) = 100 × (W f /L 3 ); Hepatosomatic index (HIS, %) = 100 × (wet weight of the hepatopancreas/W f ); Intraperitoneal ratio (IPR, %) = 100 × (intraperitoneal fat weigh/W f ); where W i and W f are the initial and final weights (g) of the fish during the experiment. t is the duration of the experiment (d); F is the weight of the feed provided to the fish; L is the average body length (cm) of the fish. 2.5 Biochemical analysis Proximate composition analyses of diets, fish whole body, and tissues were performed according to the AOAC guidelines (1984)[ 24 ]. Moisture analysis was conducted by drying the samples at 105 ℃ for 24 hours. After acid digestion with an automatic Kjeldahl System (Kjeltec 8400, FOSS, USA), the protein content of the diet and fish samples were analyzed via the Kjeltec nitrogen × 6.25 method. Moreover, the lipid and ash contents of the diets and samples were respectively analyzed with a Soxtec System (Soxtec System 2050; Foss, USA) and combustion at 550 °C for 24 h. Plasma biochemistry was characterized with a Modular P800 automatic biochemical analyzer (Roche Diagnostics, Mannheim, Germany) at Guangzhou Kingmed Center for Clinical Laboratory Co., Ltd. 2.6 Digestive enzyme activity To assess digestive enzyme activity, approximately 1 g of frozen wet heavy tissue samples (gastric, intestinal, and hepatic) were homogenized with 9 mL cold normal saline, then centrifuged at 2,500 rpm for 10 min at 4℃. The final supernatant was divided into subsamples for each enzyme analysis and stored at 4 ℃ until all the enzyme analyses were completed within 24 hours. Amylase activity was determined with a test kit (Jiancheng Biotech Co., Ltd., Nanjing, China.) according to the manufacturer's instructions. One amylase unit was defined as the activity required to hydrolyze 10 mg of starch in 30 min at 37℃. Lipase activity was determined as described by Li et al. (2005)[ 25 ] using olive oil as substrate. Each lipase activity unit was defined as the release of 1 µmol of fatty acids per minute per gram of protein. A slightly modified version of the Anson method was used to determine pepsin activity using casein as the substrate coupled with the Folin reagent, and total soluble protein was determined using bovine serum albumin as standard using the Bradford method, as described by Brito et al. (2000)[ 26 ]. 2.7 Tissues histology The collected specimens were fixed with 10% buffered formalin for at least 24 hours, then dehydrated, paraffin-embedded, and cut into 4 µm thick slices on an RM2135 rotary slicer (Wetzlar Leica, Germany). The sections were stained with hematoxylin and eosin (H&E) and histopathological examinations were performed according to standard methods. All sections were examined using a DFC495 optical microscope. 2.8 Statistical analysis All data were reported as the mean ± standard deviation (SD, n = 3). Mean values were compared using one-way ANOVA, and variance normality and heterogeneity tests were performed using SPSS19.0 (SPSS Inc., Chicago, USA). P < 0.05 was considered statistically significant. The regression model was established using Origin 9.0 (OriginLab Corporation, USA). 3 Results 3.1 Growth performance Table 2 summarizes the growth and biometry analysis results of juvenile pearl gentian groupers at different stocking densities and feeding frequencies. W f , BWG, WGR, SGR, and DFI were significantly different among the treatment groups ( P < 0.05). Specifically, W f , BWG, WGR, SGR, and DFI in treatments G1, G7, and G8 were significantly higher than those in the other treatments ( P < 0.05). G7 had the highest W f, BWG, WGR, and SGR, followed by treatments G1 and G8. Our results indicated that optimal W f , BWG, WGR, and SGR values were obtained at a stocking density of 2.2 kg m − 3 when the fish were fed twice a day, followed by a 3.3 kg m − 3 stocking density when the fish were fed 3 times a day, relatively higher W f , BWG, WGR, and SGR values were observed at a 1.1 kg m − 3 stocking density. The DFI values of juvenile hybrid groupers in the G1-G5 groups were significantly different even at high stocking densities and a 3-times-per-day feeding frequency (Table 2). The DFI in the G3 group was significantly lower than that of the other groups ( P < 0.05), whereas the highest DFI was observed in the G2 group, followed by G5. When the fish were fed twice per day, the DFI of the juvenile hybrid groupers decreased gradually with increasing stocking density. Overall, the DFI was the parameter that was most affected by stocking density and feeding frequency. No mortality was recorded throughout the experimental trials. Upon comparing the two feeding frequencies evaluated herein, no significant differences in CF were observed among groups except for G8, which exhibited a significantly higher value, and G6 had the lowest value ( P < 0.05). The HSI values of the fish in the G4 and G7 groups were significantly higher than those in the G1, G2, G6, and G9 groups ( P < 0.05) but not significantly different from those in other groups. Feeding frequency and stocking density had no significant impact on fish, except in the G1 and G10 treatment groups. According to the BWG regression model (y-axis) in response to the stocking density when the fish were fed twice a day (x-axis), the optimal stocking density was 2.004 kg/m 3 . However, based on BWG (y-axis) in response to the stocking density when the fish were fed three times a day (x-axis), the optimal stocking density was 1.343 kg/m 3 . 3.2 Biochemical analysis 3.2.1 Nutrients analyses in whole fish and muscle As the stocking density increased in fish fed three times a day, the moisture content gradually increased, and protein, fat, and ash contents were gradually reduced, particularly in the G1 group, which was significantly different from the G5 group ( P > 0.05). In contrast, increasing the stocking density in fish fed twice a day did not significantly affect moisture contents ( P > 0.05). On the other hand, protein, fat, and ash contents were significantly different between treatments, particularly in the G7 and G8 groups, which were significantly higher than the other three groups ( P 0.05). Moreover, the protein contents of G2-G5 and G9-10 were not significantly different ( P > 0.05) but were significantly lower than those of the G1 and G6-7 groups ( P 0.05) regardless of feeding frequency with increasing stocking density. The ash contents were the highest in the G1 group, followed by the G6 group, which was significantly higher than the other treatments ( P < 0.05). 3.2.2 Serum biochemical analyses Fish serum biochemical indicators are summarized in Table 4. When the stocking density was increased in fish fed three times a day, AST (aspartate aminotransferase) and CHO (cholesterol) levels increased gradually and were significantly different between the treatment groups, with the G1 group being significantly lower than the other groups ( P < 0.05). Moreover, when the fish were fed twice a day, the G7 and G8 groups were significantly lower than the other three groups ( P < 0.05). Regarding immune function, our study characterized TP (total protein), GLB (globin), ALB (albumin), GLU (glucose), LZY (lysozyme), SOD (total superoxide dismutase), and AKP (alkaline phosphatase), all of which were significantly different between treatment groups. When the fish were fed three times a day, the G4 group had the highest TP, GLB, and ALB values among all tested groups ( P < 0.05), followed by G5 and G1. Moreover, groups G1 and G2 exhibited significantly higher GLU, LZM, SOD, and AKP values than the other groups. When the fish were fed twice a day, the G7 group exhibited significantly higher TP, GLB, and LZM values than the remaining groups ( P < 0.05). The SOD and AKP values of groups G7-G9 were not significantly different, whereas G6 had the highest values. 3.3 Tissues enzyme assays As demonstrated in Table 5, there was a significant difference between the gastric, intestinal, and hepatic pepsin and lipase activity between groups ( P < 0.05). Pepsin and lipase activity increased with increasing density regardless of stocking density, with the G5 and G10 groups having the highest values ( P < 0.05), followed by G4 and G9, whereas the G1 and G6 groups had the lowest values. Additionally, no significant differences in gastric, intestinal, and hepatic pepsin and amylase activities were observed between treatments regardless of feeding frequency( P > 0.05). 3.4 Tissues histology As illustrated in Fig. 1 , the vasodilatation of the gastric wall of the G3, G4, G9, and, G10 fish indicated the presence of inflammatory cells in these organisms. Moreover, white vesicles were identified in the cells of fish in the G3, G4, G9, and G10 groups, indicating that the gastric cells in those groups were metamorphosed and became adipogenic. Additionally, gastric tissue sections of other fish groups exhibited varying degrees of inflammation and fat granules except for G7 and G8, which were normal. Figure 2 shows that increasing stocking densities in the fish fed three times a day resulted in lamina propria edemas in the intestinal tissue sections of the G3, G4, and G5 fish, as well as thinner and shorter intestinal villi, some of which were starting to detach. In contrast, the aforementioned symptoms were only identified in the G10 fish that were fed twice a day. Finally, as seen in Fig. 3 , vacuolation occurred in the G2, G5, G6, G9, and G10 fish, and nuclear deviations were observed in G3 and G4, whereas the hepatocytes in G1, G7, and G8 were normal. 4 Discussion Many factors affect fish growth, including diet, fish size, feeding time, stocking density, and feeding frequency. In this experiment, when the fish were fed three times a day, the BWG, WGR, and SGR of fish stocked at a density of 0.55 kg per tank gradually decreased with increasing stocking density and was significantly higher than the other four groups. This suggested that the growth performance of the fish in the 1.10 kg and 1.65 kg per tank stocking densities was better than that of the other groups with the same feeding frequency. In other words, feeding frequency and stocking density influenced the growth performance of this species, which was consistent with previous studies[ 27 , 28 ]. The poor growth performance observed in the high stocking density was likely due to crowding stress[ 29 ]. Additionally, our findings suggested that feeding three times per day instead of two may have affected satiety levels, thereby resulting in food waste. Several studies have reached different conclusions regarding the effect of stocking density on the growth performance of fish depending on the species. For instance, Rowland et al. (2006) [ 30 ] reported that stocking density had no significant effects on silver perch. In contrast, high stocking density decreased the final weights of Brook charr according to Vijayan et al. (1990)[ 31 ], and Boeuf et al. (1989)[ 32 ] reported that lower densities had positive effects on the growth of Atlantic salmon during smolting. According to the quadratic regression model based on the BWG of the hybrid groupers studied herein, when fed twice a day, the function was y=-0.4872163x4+6.391186x 3-28.19146x2+45.84394x-6.26（R2=1） and we predicted that the optimal stocking density was 2.004 kg/m 3 , whereas when the fish were fed three times a day, the function was y=-0.6366255x4+9.53982 x 3-50.46522x2+107.8068x-55.9（R2=1） and the optimal stocking density was 1.343 kg/m 3 . Blood can objectively reflect the nutritional metabolism of farmed animals and is an important physiological, pathological, and toxicological indicator. AST and ALT are common indicators of liver cell damage. Specifically, when liver cells are damaged, AST and ALT enzymes will infiltrate into the blood, causing a significant increase in the activity of these enzymes in the blood. Previous studies have linked the occurrence of liver damage with elevated AST and ALT levels [ 33 , 34 , 35 , 36 ]. In this study, AST levels increased significantly with stocking density, except at a 2.2 kg/m 3 . Moreover, ALT decreased significantly with increasing stocking density, particularly at 4.4 kg m − 3 and 5.5 kg m − 3 in both feeding frequencies. Our findings suggest that although increases in AST were indicative of some degree of liver damage, lower ALT levels may explain the 100% survival ratio, as it is an important indicator of fish health [ 37 ]. SOD can eliminate excess free radicals in organisms [ 38 ] and modulate the balance between the formation and elimination of free radicals. Therefore, this parameter is an important physiological indicator of antioxidant capacity and non-specific immune function. LZY and AKP are also important indicators of non-specific immunity in the body. AKP is very important for various metabolic processes including phosphorylation and dephosphorylation, as well as nutrient absorption, and its activity can be used as an indicator of fish immune function. Previous studies have demonstrated that high stocking densities were associated with decreases in SOD activity in some fish [ 39 , 40 ] and shrimps [ 41 , 42 ]. LZY is known to disrupt the structure of the bacterial cell wall, and therefore LZY levels can directly reflect lytic capacity, as well as the impact of pathogens and other environmental factors on fish health. In Senegalese sole, high stocking densities reduced plasma LZY levels[ 43 ]. A similar pattern was found in juvenile turbot[ 39 ]. In our study, high stocking densities had negative effects on SOD, AKP, and LZY concentrations, which may explain why high stocking densities often lead to diseases. Nonetheless, other studies have demonstrated that high stocking densities could increase AKP activity[ 44 , 45 ]. TG and CHO are key indicators of blood lipid levels. According to medical research, elevated TG and CHO levels are linked to fatty liver. TP includes GLB and ALB, which are involved in protein metabolism Albumin content generally accounts for more than 50% of total plasma protein content and is known to maintain osmotic pressure balance between blood vessels and tissues, mediate substance binding and transport, protect blood cells and organs, inactivating inflammation, and also acts as an antioxidant and damage repair agent, all of which are closely related to the body’s immune function. In this study, the CHO levels of groupers gradually decreased with increasing stocking density regardless of feeding frequency, meaning that higher stocking densities resulted in more liver cell damage, which would negatively affect fish digestion and fat translation. Moreover, TP and ALB showed a gradual increase, whereas GLB and GLU decreased gradually with increasing stocking density, indicating that the vital activities represented by the above indicators were gradually weakened as the stocking density increased. Previous studies have shown that the detection and analysis of digestive enzyme activities can directly reflect the physiological status of fish digestion [ 46 , 47 , 48 ]. In the present study, increases in stocking density had a positive effect on gastric, intestinal, and hepatic pepsin and lipase activities regardless of feeding frequency. However, these results were not consistent with a previous study in which the lipase, trypsin, and amylase activities of Oreochromis niloticus fingerlings were all depressed in high-density conditions (600 organisms m − 3 ) [ 44 ]. This indicated that the fish exhibited better digestive physiology at an appropriate stocking density, which may also explain why the fish had higher growth rates in the corresponding density range. However, the high stocking density in our study may have elicited crowding stress, thereby requiring additional energy expenditure to resist the stress, which in turn required an increased digestive performance. We therefore speculated that increased digestive enzyme activity may be a protective mechanism in high-density conditions; however, this hypothesis must be further explored in future studies. In this study, feeding frequency and stocking density were found to significantly influence gastric, intestinal, and hepatic tissue histology. When the fish were fed three times a day, even low stocking densities (e.g., 1.1 kg m − 3 ) resulted in pathological signs or symptoms, which was also observed in fish that were fed twice a day and stocked at 2.2 kg m − 3 and 3.3 kg m − 3 densities. These results were consistent with those of Refaey et al. (2018)[ 49 ], who reported that a high stocking density (300 fish m − 3 ) resulted in intestine and muscle histological changes. 5 Conclusions Based on observations of grouper growth performance, digestive enzyme activity, and tissue histology at different stocking densities and feeding frequencies, the optimum stocking density was 1.343 kg/m 3 (approximately 10 fish) when the fish were fed three times a day, and 2.004 kg/m 3 (approximately 20–30 fish) when the fish were fed twice a day. Abbreviations SD: stocking densities; FF: feeding frequencies; WGR: weight gain rate; SGR: specific growth rate; FER: feed efficiency ratio; PER: protein efficiency ratio; HIS: hepatosomatic index ; VSI: viscerosomatic index; IPR: intraperitoneal ratio; BWG: body weight gain; CF: condition factor; H&E; hematoxylin and eosin ; DFI: Daily feed intake; AST: aspartate aminotransferase; ALT; alanine aminotransferase; TGs: TP: total protein; GLB: globin; ALB: albumin; GLU: glucose);triglycerides; CHO: cholesterol; ALB: albumin; LZY: lysozyme; SOD: superoxide dismutase; AKP: alkaline phosphatase; AOAC: association of official analytical chemists; MS222: tricaine methanesulfonate; SD: standard deviation; W i : initial weights; W f : final weights; WW: wet weight. Declarations -Ethics approval and consent to participate All applicable international, national, and/or institutional guidelines for the care and use of animals were followed by the authors. The use of animal was approved by the Institutional Animal Care and Use Committee of Hainan Tropical Ocean University. - Consent for publication Not applicable -Availability of data and material All data generated or analysed during this study are included in this article. -Competing interests The authors declare no conflicts of interest. -Funding This study was supported by Basic and Applied basic Research Program of Hainan Province (No. 2019RC246), the Natural Science Foundation of Hainan Province (No. 20163074; No. 20163071), the Major Science and Technology Projects in Hainan Province from Science and Technology Department of Hainan Province government, China (No. ZDKJ2016009), the Hainan Key Laboratory for Conservation and Utilization of Tropical Marine Fishery Resources and Sanya Key Laboratory of Seawater Aquaculture Research (No. L1507). -Authors' contributions Yan Chen performed the conduct of Experiments, analyzed and interpreted the data regarding growth performance and biochemical analysis of this species, and was a major contributor in writing the manuscript. Huanghai & Jun Ma analyzed and explained the relevant data regarding the tissues enzyme assays and tissues histology of this fish. All authors read and approved the final manuscript. -Acknowledgements Thanks are given to the senior and junior students and teachers in College of life science and ecology,Hainan Tropical Ocean University（Sanya, China ）and Hainan Key Laboratory for Conservation and Utilization of Tropical Marine Fishery Resources, for their help in preparing the diets used in this study and assisting in sample collection. References Phillips TA, Summerfelt RC, Clayton RD. Feeding frequency effects on water quality and growth of walleye fingerlings in intensive culture. Progres Fish-Cultur. 1998;60:1–8. Wang N, Hayward RS, Noltie DB. Variation in food consumption, growth, and growth efficiency among juvenile hybrid sunfish held individually. Aquaculture. 1998;167:43–52. Booth MA, Tucker BJ, Allan GL, et al. 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B, 2004 (137):331–339. Refaey MM, Li D, Tian X, et al. High stocking density alters growth performance, blood biochemistry, intestinal histology, and muscle quality of channel catfish Ictalurus punctatus [J]. Aquaculture, 2018:S0044848617320744. Tables Table.1 Formulation and proximate composition of the experimental diet. Ingredients % Fishmeal 50.00 Soy protein concentrate 12.00 Casein 4.00 Shrimp meal 3.50 Wheat flour 20.5 Binding agents 2.00 Soybean oil 1.50 Fish oil 1.50 Squid visceral ointment 1.50 Vitamin premix 2 1.00 Mineral premix 3 1.00 Choline chloride 0.50 Monocalcium Phosphate 1.00 Proximate composition %, dry matter basis Crude protein 43.84 Crude lipid 8.20 Gross energy (MJ/kg) 17.56 Proximate composition values represent means ± standard deviation of triplicate samples, rounded to the nearest 0.1 g. Obtained from Guangzhou Rifeng Science & Technology Co., LTD (Guangzhou, China). Vitamin mixture (mg kg −1 diet): retinol acetate, 38.0; cholecalciferol, 13.2; a-tocopherol, 210.0; thiamin, 115.0; riboflavin, 380.0 pyridoxine 88.0; pantothenic acid, 368.0; niacin acid, 1030.0 biotin, 10.0; folic acid, 20.0; vitamin B12, 1.3; inositol, 4000.0; ascorbic acid, 500.0 (Ding et al., 2010). Mineral mixture (mg kg −1 diet): MgSO 4 ·7H 2 O, 3568.0; NaH 2 PO 4 ·2H 2 O, 25568.0; KCl, 3020.5; KAl (SO 4 ) 2 , 8.3; CoCl2, 28.0; ZnSO 4 ·7H 2 O, 353.0; Calactate, 15968.0; CuSO 4 ·5H 2 O, 9.0; KI, 7.0; MnSO 4 ·4H 2 O, 63.1; Na 2 SeO 3 , 1.5; C 6 H 5 O 7 Fe·5H 2 O, 1533.0; NaCl, 100.0; NaF, 4.0 (Ding et al., 2010). Table.2 Growth and biometry of juvenile pearl gentian groupers (mean ± S.D., n=3) Group Growth performance Biometric indices W i (g) W f (g) BWG(g) WGR (%) SGR(%/d) DFI (%/d) Survival(%) CF(g/cm 3 ) HIS (%) IPR (%) G1 11.03±0.09 28.88±0.003b 17.85±0.11b 1.62±0.02b 2.29±0.02b 2.65±0.01d 100.00±0.00 1.53±0.16bcd 1.83±0.50c 0.89±0.52b G2 11.10±0.05 24.89±0.02d 14.79±0.03c 0.89±0.04d 2.15±0.01cd 2.91±0.04a 100.00±0.00 1.67±0.24abc 1.98±0.84bc 1.27±0.61ab G3 11.22±0.18 21.14±0.06f 9.92±0.59f 0.65±0.04e 1.51±0.08g 2.23±0.01e 100.00±0.00 1.56±0.23abcd 2.72±0.83ab 1.12±0.57ab G4 11.28±0.10 22.76±0.12ef 11.48±0.12e 0.85±0.03d 1.67±0.02f 2.73±0.02c 100.00±0.00 1.76±0.37ab 2.87±0.87a 1.24±0.26ab G5 11.02±0.07 21.62±0.02f 10.59±0.06f 0.73±0.01e 1.60±0.01fg 2.82±0.03b 100.00±0.00 1.72±0.29abc 2.13±0.64abc 1.30±0.75ab G6 11.03±0.04 23.96±0.06de 13.39±0.28d 1.27±0.06c 1.95±0.06e 2.74±0.01c 100.00±0.00 1.38±0.16d 1.53±0.86c 0.97±0.48ab G7 11.12±0.06 33.86±0.21a 23.69±0.11a 2.33±0.01a 2.86±0.01a 2.67±0.01d 100.00±0.00 1.67±0.15abc 2.95±0.62a 1.28±0.52ab G8 11.17±0.07 30.18±1.72b 17.63±0.38b 1.29±0.02c 2.26±0.04bc 2.10±0.01f 100.00±0.00 1.80±0.24a 2.20±0.90abc 1.41±0.67ab G9 11.32±0.07 26.79±0.08c 15.47±0.08c 1.26±0.01c 2.05±0.01de 1.96±0.01g 100.00±0.00 1.49±0.24cd 2.00±0.54bc 1.10±0.55ab G10 11.17±0.03 26.64±0.22c 15.10±0.36c 1.24±0.03c 1.99±0.05e 1.86±0.01h 100.00±0.00 1.58±0.15abcd 2.80±0.73ab 1.65±0.85a ANOVA ( P value) Breeding density < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 0.047 0.012 0.150 Feeding frequency < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 0.221 0.948 0.375 Interactions < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 0.032 0.003 0.763 Values (mean ± S.D.) with different letters within the same row are significantly different ( P <0.05). The absence of letters indicates no significant difference between treatments. ANOVA: Two-way analysis of variance Table 3 Proximate composition of muscle and whole body of juvenile pearl gentian groupers (mean ± S.D., n=3). Group Proximate composition of muscle (% WW a ) Proximate composition of whole body(% WW) Moisture Protein Fat Ash Moisture Protein Fat Ash G1 75.70±0.33de 20.05±0.37abc 1.34±0.06bcd 1.53±0.01c 67.28±0.13e 19.53±0.11a 6.00±0.42ab 6.77±0.10a G2 77.32±0.11bcd 18.71±0.13cd 1.31±0.06cde 1.54±0.01b 71.64±0.07ab 17.78±0.04b 5.06±0.46ab 5.32±0.01d G3 78.69±0.21ab 17.20±0.20e 1.16±0.07ef 1.46±0.01e 71.21±0.12b 18.12±0.07b 4.05±0.06b 5.28±0.01d G4 78.47±0.26ab 17.65±0.23de 1.43±0.03bc 1.46±0.01e 71.73±0.19ab 18.21±0.25b 4.23±0.06ab 4.84±0.01f G5 79.85±0.39a 16.36±0.29e 1.39±0.05bcd 1.33±0.01g 71.24±0.09b 18.23±0.25b 4.77±0.05ab 5.08±0.03e G6 77.48±1.54bc 19.06±1.31bc 1.03±0.07f 1.49±0.01d 66.94±0.23e 20.65±0.30a 7.49±1.87ab 6.38±0.03b G7 76.51±0.23cde 20.28±0.32ab 1.74±0.03a 1.44±0.01f 68.00±0.20d 20.61±0.16a 5.41±0.07ab 5.49±0.05c G8 75.18±0.21e 21.05±0.10a 1.48±0.06b 1.54±0.01b 68.07±0.21d 20.93±0.19a 8.58±3.78a 5.32±0.03d G9 76.22±0.17cde 20.37±0.13ab 1.38±0.02bcd 1.57±0.01a 71.78±0.19a 18.07±0.47b 4.37±0.07ab 4.98±0.01e G10 77.25±0.18bcd 19.68±0.16abc 1.24±0.04de 1.46±0.01e 70.56±0.22c 18.12±0.12b 4.36±0.06ab 5.25±0.01d ANOVA( P value) Breeding density 0.005 0.012 < 0.001 < 0.001 < 0.001 < 0.001 0.295 < 0.001 Feeding frequency < 0.001 < 0.001 0.139 < 0.001 < 0.001 < 0.001 0.157 0.250 Interactions < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 0.377 < 0.001 Values (mean ± S.D.) with different letters within the same row are significantly different (P<0.05). The absence of letters indicates no significant difference between treatments. a WW: wet weight. Table.4 Serum biochemistry (mean ± S.D., n=3). Group Items AST ALT TP GLB ALB TG CHO GLU LZM SOD AKP G1 17.33±0.58h 269.00±8.39a 30.50±0.06e 21.03±0.12bc 9.47±0.07f 0.99±0.01fg 1.65±0.01h 22.60±0.08b 91.41±4.82e 4.46±0.09a 14.53±0.13a G2 52.33±0.58d 152.00±3.46b 26.97±0.09h 17.27±0.13f 9.70±0.06ef 1.31±0.02c 1.87±0.04g 20.45±0.16c 76.67±5.09f 4.73±0.14c 13.80 ±0.03b G3 33.00±1.00f 138.33±6.57b 29.37±0.15fg 19.37±0.09d 10.00±0.10e 0.90±0.03h 2.38±0.02f 9.73±0.04e 58.89±4.84gh 4.51±0.06a 13.33±0.10c G4 53.33±0.58d 52.67±1.76c 36.87±0.17a 21.53±0.07a 15.33±0.15a 1.03±0.03ef 2.76±0.01d 8.57±0.04e 53.33±3.33h 4.35±0.03a 12.37 ±0.08d G5 60.00±0.00c 47.67±2.33c 31.30±0.10d 17.17±0.07f 14.13±0.15b 0.78±0.03i 2.63±0.01e 12.70±0.10d 66.67±0.00fg 4.01±0.13b 10.54±0.09f G6 21.00±1.00g 267.67±2.03a 29.07±0.03g 20.67±0.15c 8.40±0.15g 1.53±0.02b 1.44±0.03i 2.05±0.03f 166.67±0.00c 4.00±0.11b 11.50 ±0.06e G7 39.00±1.73e 258.33±9.94a 33.20±0.15b 21.97±0.07a 8.23±0.19g 0.93±0.03gh 3.56±0.03b 13.63±0.19d 285.55±5.37a 3.77±0.06bc 7.38 ±0.08g G8 18.00±1.00h 108.67±5.04b 29.73±0.23f 21.43±0.35ab 8.30±0.15g 1.65±0.01a 3.37±0.01c 38.37±1.39a 201.11±1.92b 3.76±0.02bc 7.15 ±0.05g G9 69.67±1.15b 36.67±0.33c 30.70±0.26e 18.43±0.18e 12.27±0.09d 1.08±0.02e 2.78±0.01d 22.87±0.08b 277.78±9.62a 3.57±0.02c 7.17 ±0.03g G10 77.33±0.58a 43.67±1.33c 32.10±0.10c 18.83±0.09e 13.27±0.12c 1.19±0.03d 4.10±0.07a 22.55±0.24b 108.33±8.34d 1.56±0.04d 7.58 ±0.02f ANOVA( P value) Breeding density < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 Feeding frequency < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 Interactions < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 Values (mean ± S.D.) with different letters within the same row are significantly different ( P <0.05). The absence of letters indicates no significant difference between treatments. a WW: wet weight. AST: Aspartate aminotransferase; ALT: Alanine transaminase; TP: Total protein; GLB: Globulin; ALB: Albumin; TG: Triglyceride; CHO: Cholesterol; GLU: Glucose; LZY: Serum lysozyme; SOD: Superoxide dismutase; AKP: Alkaline phosphatase Table.5 Digestive enzyme activities (mean ± S.D., n=3). Group Gastric Intestinal Hepatic Pepsin (U/mg prot) Amylase (U/mg prot) Lipase (U/mg prot) Pepsin (U/mg prot) Amylase (U/mg prot) Lipase (U/mg prot) Pepsin (U/mg prot) Amylase (U/mg prot) Lipase (U/mg prot) G1 17.23 0.68±0.00 18.08±0.12f 1.54±0.01d 0.26±0.00 30.61±0.13ef 4.66±0.01d 0.10±0.00 2.23±0.04d G2 18.48 0.71±±0.00 19.11±0.11e 1.62±0.01c 0.25±0.00 31.76±0.11d 5.03±0.01cd 0.11±0.00 2.56±0.04cd G3 20.67 0.77±0.00 20.0±0.14ad 1.68±0.01bc 0.24±0.00 32.33±0.16c 5.37±0.05bc 0.12±0.00 2.65±0.08c G4 22.32 0.80±0.00 20.71±0.21ab 1.76±0.02ab 0.23±0.00 33.07±0.20b 5.44±0.00bc 0.12±0.00 2.90±0.03ab G5 24.21 0.84±0.00 21.18±0.25a 1.78±0.01ab 0.22±0.00 34.80±0.12a 5.94±0.01b 0.13±0.00 2.97±0.05a G6 17.38 0.70±0.00 17.63±0.15f 1.57±0.02cd 0.28±0.00 30.33±0.21f 4.87±0.01dc 0.10±0.00 2.12±0.04e G7 19.48 0.74±0.00 18.65±0.20e 1.63±0.01c 0.23±0.00 30.85±0.10e 5.19±0.01c 0.11±0.00 2.24±0.06d G8 22.57bc 0.80±0.00 19.65±0.19d 1.72±0.01b 0.22±0.00 31.42±0.09d 5.61±0.01cd 0.11±0.00 2.68±0.06c G9 23.62 0.83±0.00 19.84±0.16d 1.77±0.01ab 0.22±0.00 32.23±0.12c 5.88±0.01bc 0.12±0.00 2.79±0.06b G10 25.03 0.86±0.00 20.61±0.25bc 1.80±0.02a 0.21±0.00 33.21±0.12b 6.24±0.00a 0.14±0.00 2.88±0.04ab ANOVA(Pvalue) Breeding density < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 Feeding frequency < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 Interactions < 0.001 0.136 0.734 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 Values (mean ± S.D.) with different letters within the same row are significantly different ( P <0.05). The absence of letters indicates no significant difference between treatments. a WW: wet weight. Supplementary Files AuthorChecklistFull.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-103762","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research article","associatedPublications":[],"authors":[{"id":4481191,"identity":"fac730e3-ee28-4e82-9d9e-d781d98b28dc","order_by":0,"name":"Yan Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA30lEQVRIiWNgGAWjYFAC5oMPPvywkTNgYD5w4EMFUVrYkg1n9qQZGzCwJR6ccYYoLTxmwjxshxM3MPAYH+ZtIUID/4wcM2YensPG5tI9Hw7wNjDI84sdwK9F4kZa2cM5FulylnPObjgguYPBcObsBPxaDCSStxu84bE2NriRu+GA4RmGBIPbBLUkmEnwsDEnbriR8+BAYhtRWlLMJHnYnEFaGA4cJEaLxJln0EC+kWZwsOGMBGG/8LcnQ6PyRvLjz38qbOT5pQloYRBAVSBBQDnYmgNEKBoFo2AUjIKRDQDrAE1KoHCHwwAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-9922-0489","institution":"Hainan Tropical Ocean University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Chen","suffix":""},{"id":4481192,"identity":"1d380a71-436a-4bf4-b1b7-0ddfc730f4ae","order_by":1,"name":"Hai Huang","email":"","orcid":"","institution":"Hainan Tropical Ocean University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hai","middleName":"","lastName":"Huang","suffix":""},{"id":4481193,"identity":"d5cfc6eb-e3d4-415b-878b-c6ed53bdcb6e","order_by":2,"name":"Jun Ma","email":"","orcid":"","institution":"Hainan Tropical Ocean University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Ma","suffix":""},{"id":4481194,"identity":"59b32d45-4c41-47ad-9f10-4c4f5eed6b08","order_by":3,"name":"Wenkan Liu","email":"","orcid":"","institution":"Hainan Tropical Ocean University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wenkan","middleName":"","lastName":"Liu","suffix":""},{"id":4481195,"identity":"5d3ee5eb-ab26-4777-9c9b-771b79572d51","order_by":4,"name":"Honggan Zhong","email":"","orcid":"","institution":"Hainan Tropical Ocean University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Honggan","middleName":"","lastName":"Zhong","suffix":""},{"id":4481196,"identity":"0a339e6c-a32b-42b0-8295-08b00d217b26","order_by":5,"name":"Jiechao Zhang","email":"","orcid":"","institution":"Hainan Tropical Ocean University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jiechao","middleName":"","lastName":"Zhang","suffix":""},{"id":4481197,"identity":"63b8724d-b6ba-4870-98a0-aab12be3c6c0","order_by":6,"name":"Jiaohao Zhong","email":"","orcid":"","institution":"Hainan Tropical Ocean University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jiaohao","middleName":"","lastName":"Zhong","suffix":""},{"id":4481198,"identity":"e5ee5b09-a9c9-4927-9e00-f552eed80eac","order_by":7,"name":"Feng Ye","email":"","orcid":"","institution":"Hainan Tropical Ocean University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Feng","middleName":"","lastName":"Ye","suffix":""},{"id":4481199,"identity":"94027102-1b23-4d2c-96ca-026c2fd0851f","order_by":8,"name":"Chengcai Li","email":"","orcid":"","institution":"Hainan Tropical Ocean University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chengcai","middleName":"","lastName":"Li","suffix":""},{"id":4481200,"identity":"2aa581b7-e48f-47fa-ad57-f41c67d3dabe","order_by":9,"name":"Xiaomei Wang","email":"","orcid":"","institution":"Hainan Tropical Ocean University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiaomei","middleName":"","lastName":"Wang","suffix":""},{"id":4481201,"identity":"08409703-7a55-443d-af0a-05adc149660b","order_by":10,"name":"Chenxi Xu","email":"","orcid":"","institution":"Hainan Tropical Ocean University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chenxi","middleName":"","lastName":"Xu","suffix":""},{"id":4481202,"identity":"4326565a-bf8b-4867-9614-b37f8824063f","order_by":11,"name":"Jiajie Zhu","email":"","orcid":"","institution":"Hainan Tropical Ocean University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jiajie","middleName":"","lastName":"Zhu","suffix":""}],"badges":[],"createdAt":"2020-11-05 23:56:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-103762/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-103762/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":3539830,"identity":"85c30245-e6b8-4d5f-8251-597ca07d5f21","added_by":"auto","created_at":"2020-11-12 16:46:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":260876,"visible":true,"origin":"","legend":"H\u0026E stained gastric tissue sections of juvenile pearl gentian grouper. \n(A-J) Gastric tissue sections of fish in groups G1 to G10, respectively \n(H\u0026E, 400)\n","description":"","filename":"Onlinefloatimage1.Png","url":"https://assets-eu.researchsquare.com/files/rs-103762/v1/f5f2283f8f6fab4925cf807f.Png"},{"id":3539831,"identity":"b0e09488-5108-4225-8c92-ea878570e36b","added_by":"auto","created_at":"2020-11-12 16:46:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":246745,"visible":true,"origin":"","legend":"H\u0026E stained intestinal tissue sections of juvenile pearl gentian grouper.\n(A-J) Intestinal tissue sections of fish in groups G1 to G10, respectively \n(H\u0026E, 400)\n","description":"","filename":"Onlinefloatimage2.Png","url":"https://assets-eu.researchsquare.com/files/rs-103762/v1/55fdbd1d3a24a0760f5cab0b.Png"},{"id":3539832,"identity":"04e2edb5-eeca-4504-95e0-af877ad542de","added_by":"auto","created_at":"2020-11-12 16:46:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":310290,"visible":true,"origin":"","legend":" H\u0026E stained hepatic tissue sections of juvenile pearl gentian grouper.\n(A-J) Hepatic tissue sections of fish in groups G1 to G10, respectively (H\u0026E, 400)","description":"","filename":"Onlinefloatimage3.Png","url":"https://assets-eu.researchsquare.com/files/rs-103762/v1/0b193e4baea65415c806d962.Png"},{"id":3539833,"identity":"39dcc1f9-821a-4975-ad58-996e9a41986b","added_by":"auto","created_at":"2020-11-12 16:46:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":90958,"visible":true,"origin":"","legend":"Quadratic regression model based on body weight gain (y-axis) in response to fish stocking density when fed twice a day (x-axis).","description":"","filename":"4.PNG","url":"https://assets-eu.researchsquare.com/files/rs-103762/v1/8b1cdef45e7a34580fb4fc10.PNG"},{"id":3539834,"identity":"18ebb94a-3982-45f4-9bad-06a0643890e5","added_by":"auto","created_at":"2020-11-12 16:46:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":76198,"visible":true,"origin":"","legend":"Quadratic regression model based on body weight gain (y-axis) in response to fish stocking density when fed three times a day (x-axis).","description":"","filename":"5.PNG","url":"https://assets-eu.researchsquare.com/files/rs-103762/v1/5b9ca4762a7f6c90a56e0314.PNG"},{"id":13613438,"identity":"5cddd38f-e2ef-4027-8f8c-8b520cab342b","added_by":"auto","created_at":"2021-09-17 06:37:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3827632,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-103762/v1/2fcb46d7-ad7d-4326-b21b-bec8f4a66939.pdf"},{"id":3539835,"identity":"ab49faa2-ff46-4f76-bb12-b577fe057437","added_by":"auto","created_at":"2020-11-12 16:46:28","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":172188,"visible":true,"origin":"","legend":"","description":"","filename":"AuthorChecklistFull.pdf","url":"https://assets-eu.researchsquare.com/files/rs-103762/v1/137b4d3d59c01e30c5be5b38.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eEffects of\u0026nbsp;Stocking\u0026nbsp;Density and FeedingfFrequency on the Growth Performance,\u0026nbsp;Digestive Enzyme Activity, and Tissues Histology of\u0026nbsp;Hybrid Grouper (Epinephelus Fuscoguttatus♀×E. Lanceolatus♂)\u003c/p\u003e","fulltext":[{"header":"Background","content":" \u003cp\u003eThe balance between rapid fish growth, feed utilization efficiency, feeding strategy, and intensive fish-farming practices is a key point in fish aquaculture [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Feeding time and frequency have been reported to affect feed intake and growth performance in different fish species including the Australian snapper \u003cem\u003ePagrus auratus\u003c/em\u003e [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], pikeperch \u003cem\u003eSander lucioperca\u003c/em\u003e [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], African catfish \u003cem\u003eClariasgariepinus, Burchell 1822\u003c/em\u003e [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], flounder \u003cem\u003ePlatichthysflesusluscus\u003c/em\u003e [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] and rock bream \u003cem\u003eOplegnathusfasciatus\u003c/em\u003e [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Water quality, management procedures, stocking density, feeding frequency, and nutritional conditions are all directly associated with fish stress [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFeeding frequency is a well-known regulator of growth performance, feed intake, and chemical composition of fish, and therefore optimizing this factor can substantially reduce aquaculture production costs and prevent water quality deterioration resulting from excess feeding. In practice, fish are fed by most farmers to satiety based on visual observation. However, it is very difficult to identify the point at which fish are satiated and fish are overfed frequently. The stomach and intestine may exceed their capacity as a result of overfeeding, which leads to decreases in digestive efficiency and feed utilization [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. On the other hand, an insufficient feeding frequency leads to poor growth and high mortality, particularly in intensive systems[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. For example, sporadic and low feeding rates may lead to slower growth rates, increased hunger, intraspecies aggression, and increased cannibalism [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIntensive fish farming practices often result in fish stress and poor health. In contrast, improved feeding conditions not only benefit the welfare of fish but also aquaculture profits [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In most fish species, high stocking densities adversely affect fish survival, weight gain, and specific growth rates[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. These effects are attributed to the competition for food and/or space resulting from food scarcity [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], which leads to the establishment of hierarchy [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Additionally, high stocking densities can increase the likelihood of physical injuries, stress, and susceptibility, and can also modulate swimming and aggressive behaviors[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. There is considerable evidence of welfare decline in rainbow trout \u003cem\u003eOncorhynchus mykiss\u003c/em\u003e stocked at high densities; however, excessive aggression and poor feeding rates may also occur when rainbow trout are reared at very low densities [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHybrid grouper (\u003cem\u003eEpinephelus fuscoguttatus♀\u0026times;E. lanceolatus♂\u003c/em\u003e) is among the most widely farmed fish species worldwide, particularly in China. Although some work has been done regarding the effects of temperature, nutrients, and disease on grouper growth [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], studies on the effect of feeding frequency and stocking density on this species in Chinese waters remains scarce. Moreover, the synergistic effects of both parameters have not been characterized. Therefore, our study sought to evaluate the effects of stocking density and feeding frequency on growth performance, body composition, digestive enzyme activity, and histology of juvenile hybrid groupers, with the overarching goal of determining whether these two stressful fish farming practices affect fish physiology synergistically or individually.\u003c/p\u003e "},{"header":"2 Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section3\"\u003e \u003ch2\u003e2.1 Experimental diets\u003c/h2\u003e \u003cp\u003eDiet formulation and proximate analysis [Association of Official Analytical Chemists (AOAC), 1984][\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] details are provided in Table\u0026nbsp;1. The diet ingredients including fishmeal, soy protein concentrate, casein, shrimp meal, wheat flour, binding agents, soybean oil, fish oil, squid visceral ointment, choline chloride, and monocalcium phosphate were acquired from Guangzhou Rifeng Science \u0026amp; Technology Co., LTD. The mineral and vitamin premixes were purchased from Guangzhou Ashare Aquatech Co., Ltd\u003c/p\u003e \u003cp\u003eAll dry ingredients were first thoroughly mixed in a blender, after which the oil was slowly added to the mixture while blending. Deionized water (300\u0026nbsp;mL kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e dry mixture) was then slowly mixed in to achieve a dough-like consistency. This dough was then extruded into pellets (3\u0026nbsp;mm in diameter) using a twin-screw extruder (South China University of Technology F-26 (II)) and dried in a 60 ℃ oven until the moisture content was less than 8%. All pellets were sealed in plastic bags and frozen (\u0026minus;\u0026thinsp;20 \u0026deg; C) until used.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Experimental fish and feeding trial\u003c/h2\u003e \u003cp\u003ePrior to the experiment, the use of animals was approved by the \"Institutional Animal Care and Use Committee of Hainan Tropical Ocean University\" and \"Hainan Key Laboratory for Conservation and Utilization of Tropical Marine Fishery Resources\" (20161111A1). Juvenile hybrid groupers were acquired from a fish farm (Hainan Chenhai Products Co., Ltd.). The fish were originally kept in a high-quality well water circulation system, after which they were randomly assigned to 30 outdoor circular tanks (500 Litres) that were continuously supplied with 30 liters of water per minute. The following were the inflow water quality parameters: 27.0\u0026ndash;28.9 ℃; 7.0\u0026ndash;8.2\u0026nbsp;mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of dissolved oxygen; pH 6.8\u0026ndash;7.5; 28.1\u0026ndash;31.1\u0026permil; salinity; and 0.05\u0026ndash;0.1\u0026nbsp;mg/L total ammonia nitrogen. Feed intake and fish mortality were recorded daily.\u003c/p\u003e \u003cp\u003eTwo variables were considered together: feeding frequencies (FF) [3 meals a day (FF3) and 2 meals a day (FF2)] and stocking density (SD) [1.1\u0026nbsp;kg m\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e (0.55\u0026nbsp;kg fish per tanks, SD1.1), 2.2\u0026nbsp;kg m\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e (1.1\u0026nbsp;kg fish per tank, SD2.2), 3.3\u0026nbsp;kg m\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e (1.65\u0026nbsp;kg fish per tank, SD3.3), 4.4\u0026nbsp;kg m\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e (2.2\u0026nbsp;kg fish per tank, SD4.4), and 5.5\u0026nbsp;kg m\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e(2.75\u0026nbsp;kg fish per tank, CD5.5)]. Based on this 5\u0026thinsp;\u0026times;\u0026thinsp;2 factorial design, ten treatments were analyzed in triplicate randomly: G1(SD1.1,FF3), G2(SD2.2,FF3), G3(SD3.3,FF3), G4(SD4.4,FF3), G5(SD5.5,FF3), G6(SD1.1,FF2), G7(SD2.2,FF2), G8(SD3.3,FF2), G9(SD4.4,FF2), and G10(SD5.5,FF2). The trial was conducted for 42 days without counting a 15-day pre-trial acclimation period.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Sample collection\u003c/h2\u003e \u003cp\u003eAt the end of the growth test, the fish were collected after 24 hours of fasting, anesthetized with tricaine methanesulfonate (MS222) (50\u0026nbsp;mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), and weighed/measured. Fish numbers and weights per tank were recorded to calculate the feed efficiency ratio (FER), specific growth rate (SGR), survival, and protein efficiency ratio (PER). Eight fish were randomly selected from each tank for proximate analysis, three for whole-body composition tests and five for white muscle components.\u003c/p\u003e \u003cp\u003eThe viscera, liver, and intraperitoneal fat of fish were quickly removed and weighed to calculate hepatosomatic index (HSI), viscerosomatic index (VSI), and intraperitoneal ratio (IPR). Blood samples were taken from the tail vein of eight fish in each tank with syringes and centrifuged at 3000\u0026nbsp;g for 10 minutes at 4 ℃.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Evaluation of growth performance\u003c/h2\u003e \u003cp\u003eThe main growth performance variables were calculated as follows:\u003c/p\u003e \u003cp\u003eWG (%)\u0026thinsp;=\u0026thinsp;100 \u0026times; (final mean weight - initial mean weight)/initial mean weight (g)\u003c/p\u003e \u003cp\u003eSurvival (%)\u0026thinsp;=\u0026thinsp;100\u0026thinsp;\u0026times;\u0026thinsp;final fish number/initial fish number\u003c/p\u003e \u003cp\u003eAverage body weight gain (BWG, g) = (W\u003csub\u003ef\u003c/sub\u003e\u0026minus; W\u003csub\u003ei\u003c/sub\u003e)/number of fishes;\u003c/p\u003e \u003cp\u003eSpecific growth ratio (SGR, % \u003csup\u003ed\u0026minus;1\u003c/sup\u003e)\u0026thinsp;=\u0026thinsp;100 \u0026times; (In Wf\u0026thinsp;\u0026minus;\u0026thinsp;Wi)/t;\u003c/p\u003e \u003cp\u003eWeight gain ratio (WGR) = (W\u003csub\u003ef\u003c/sub\u003e\u0026minus; W\u003csub\u003ei\u003c/sub\u003e)/W\u003csub\u003ei\u003c/sub\u003e;\u003c/p\u003e \u003cp\u003eDaily feed intake (DFI, % d\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u0026thinsp;=\u0026thinsp;100\u0026thinsp;\u0026times;\u0026thinsp;dry feed intake/[(initial feed weight\u0026thinsp;+\u0026thinsp;final weight)/2\u0026thinsp;\u0026times;\u0026thinsp;t];\u003c/p\u003e \u003cp\u003eCondition factor (CF, g cm\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e)\u0026thinsp;=\u0026thinsp;100 \u0026times; (W\u003csub\u003ef\u003c/sub\u003e/L\u003csup\u003e3\u003c/sup\u003e);\u003c/p\u003e \u003cp\u003eHepatosomatic index (HIS, %)\u0026thinsp;=\u0026thinsp;100 \u0026times; (wet weight of the hepatopancreas/W\u003csub\u003ef\u003c/sub\u003e);\u003c/p\u003e \u003cp\u003eIntraperitoneal ratio (IPR, %)\u0026thinsp;=\u0026thinsp;100 \u0026times; (intraperitoneal fat weigh/W\u003csub\u003ef\u003c/sub\u003e);\u003c/p\u003e \u003cp\u003ewhere W\u003csub\u003ei\u003c/sub\u003e and W\u003csub\u003ef\u003c/sub\u003e are the initial and final weights (g) of the fish during the experiment. t is the duration of the experiment (d); F is the weight of the feed provided to the fish; L is the average body length (cm) of the fish.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Biochemical analysis\u003c/h2\u003e \u003cp\u003eProximate composition analyses of diets, fish whole body, and tissues were performed according to the AOAC guidelines (1984)[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Moisture analysis was conducted by drying the samples at 105 ℃ for 24 hours. After acid digestion with an automatic Kjeldahl System (Kjeltec 8400, FOSS, USA), the protein content of the diet and fish samples were analyzed via the Kjeltec nitrogen\u0026thinsp;\u0026times;\u0026thinsp;6.25 method. Moreover, the lipid and ash contents of the diets and samples were respectively analyzed with a Soxtec System (Soxtec System 2050; Foss, USA) and combustion at 550\u0026nbsp;\u0026deg;C for 24\u0026nbsp;h.\u003c/p\u003e \u003cp\u003ePlasma biochemistry was characterized with a Modular P800 automatic biochemical analyzer (Roche Diagnostics, Mannheim, Germany) at Guangzhou Kingmed Center for Clinical Laboratory Co., Ltd.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Digestive enzyme activity\u003c/h2\u003e \u003cp\u003eTo assess digestive enzyme activity, approximately 1\u0026nbsp;g of frozen wet heavy tissue samples (gastric, intestinal, and hepatic) were homogenized with 9\u0026nbsp;mL cold normal saline, then centrifuged at 2,500\u0026nbsp;rpm for 10\u0026nbsp;min at 4℃. The final supernatant was divided into subsamples for each enzyme analysis and stored at 4 ℃ until all the enzyme analyses were completed within 24 hours. Amylase activity was determined with a test kit (Jiancheng Biotech Co., Ltd., Nanjing, China.) according to the manufacturer's instructions. One amylase unit was defined as the activity required to hydrolyze 10\u0026nbsp;mg of starch in 30\u0026nbsp;min at 37℃. Lipase activity was determined as described by Li et al. (2005)[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] using olive oil as substrate. Each lipase activity unit was defined as the release of 1\u0026nbsp;\u0026micro;mol of fatty acids per minute per gram of protein. A slightly modified version of the Anson method was used to determine pepsin activity using casein as the substrate coupled with the Folin reagent, and total soluble protein was determined using bovine serum albumin as standard using the Bradford method, as described by Brito et al. (2000)[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Tissues histology\u003c/h2\u003e \u003cp\u003eThe collected specimens were fixed with 10% buffered formalin for at least 24 hours, then dehydrated, paraffin-embedded, and cut into 4\u0026nbsp;\u0026micro;m thick slices on an RM2135 rotary slicer (Wetzlar Leica, Germany). The sections were stained with hematoxylin and eosin (H\u0026amp;E) and histopathological examinations were performed according to standard methods. All sections were examined using a DFC495 optical microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Statistical analysis\u003c/h2\u003e \u003cp\u003eAll data were reported as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD, n\u0026thinsp;=\u0026thinsp;3). Mean values were compared using one-way ANOVA, and variance normality and heterogeneity tests were performed using SPSS19.0 (SPSS Inc., Chicago, USA). \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. The regression model was established using Origin 9.0 (OriginLab Corporation, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":" \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e3.1 Growth performance\u003c/h2\u003e \u003cp\u003eTable\u0026nbsp;2 summarizes the growth and biometry analysis results of juvenile pearl gentian groupers at different stocking densities and feeding frequencies. W\u003csub\u003ef\u003c/sub\u003e, BWG, WGR, SGR, and DFI were significantly different among the treatment groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Specifically, W\u003csub\u003ef\u003c/sub\u003e, BWG, WGR, SGR, and DFI in treatments G1, G7, and G8 were significantly higher than those in the other treatments (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). G7 had the highest W\u003csub\u003ef,\u003c/sub\u003e BWG, WGR, and SGR, followed by treatments G1 and G8. Our results indicated that optimal W\u003csub\u003ef\u003c/sub\u003e, BWG, WGR, and SGR values were obtained at a stocking density of 2.2\u0026nbsp;kg m\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e when the fish were fed twice a day, followed by a 3.3\u0026nbsp;kg m\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e stocking density when the fish were fed 3 times a day, relatively higher W\u003csub\u003ef\u003c/sub\u003e, BWG, WGR, and SGR values were observed at a 1.1\u0026nbsp;kg m\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e stocking density.\u003c/p\u003e \u003cp\u003eThe DFI values of juvenile hybrid groupers in the G1-G5 groups were significantly different even at high stocking densities and a 3-times-per-day feeding frequency (Table\u0026nbsp;2). The DFI in the G3 group was significantly lower than that of the other groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), whereas the highest DFI was observed in the G2 group, followed by G5. When the fish were fed twice per day, the DFI of the juvenile hybrid groupers decreased gradually with increasing stocking density. Overall, the DFI was the parameter that was most affected by stocking density and feeding frequency. No mortality was recorded throughout the experimental trials.\u003c/p\u003e \u003cp\u003eUpon comparing the two feeding frequencies evaluated herein, no significant differences in CF were observed among groups except for G8, which exhibited a significantly higher value, and G6 had the lowest value (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The HSI values of the fish in the G4 and G7 groups were significantly higher than those in the G1, G2, G6, and G9 groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) but not significantly different from those in other groups. Feeding frequency and stocking density had no significant impact on fish, except in the G1 and G10 treatment groups.\u003c/p\u003e \u003cp\u003eAccording to the BWG regression model (y-axis) in response to the stocking density when the fish were fed twice a day (x-axis), the optimal stocking density was 2.004\u0026nbsp;kg/m\u003csup\u003e3\u003c/sup\u003e. However, based on BWG (y-axis) in response to the stocking density when the fish were fed three times a day (x-axis), the optimal stocking density was 1.343\u0026nbsp;kg/m\u003csup\u003e3\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Biochemical analysis\u003c/h2\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e3.2.1 Nutrients analyses in whole fish and muscle\u003c/h2\u003e \u003cp\u003eAs the stocking density increased in fish fed three times a day, the moisture content gradually increased, and protein, fat, and ash contents were gradually reduced, particularly in the G1 group, which was significantly different from the G5 group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). In contrast, increasing the stocking density in fish fed twice a day did not significantly affect moisture contents (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). On the other hand, protein, fat, and ash contents were significantly different between treatments, particularly in the G7 and G8 groups, which were significantly higher than the other three groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eWhen the stocking density was increased in fish fed three times a day, the moisture content of the whole fish was no significantly different (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Moreover, the protein contents of G2-G5 and G9-10 were not significantly different (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) but were significantly lower than those of the G1 and G6-7 groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). There was no significant difference in fat content (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) regardless of feeding frequency with increasing stocking density. The ash contents were the highest in the G1 group, followed by the G6 group, which was significantly higher than the other treatments (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2.2 Serum biochemical analyses\u003c/h2\u003e \u003cp\u003eFish serum biochemical indicators are summarized in Table\u0026nbsp;4. When the stocking density was increased in fish fed three times a day, AST (aspartate aminotransferase) and CHO (cholesterol) levels increased gradually and were significantly different between the treatment groups, with the G1 group being significantly lower than the other groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Moreover, when the fish were fed twice a day, the G7 and G8 groups were significantly lower than the other three groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eRegarding immune function, our study characterized TP (total protein), GLB (globin), ALB (albumin), GLU (glucose), LZY (lysozyme), SOD (total superoxide dismutase), and AKP (alkaline phosphatase), all of which were significantly different between treatment groups. When the fish were fed three times a day, the G4 group had the highest TP, GLB, and ALB values among all tested groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), followed by G5 and G1. Moreover, groups G1 and G2 exhibited significantly higher GLU, LZM, SOD, and AKP values than the other groups. When the fish were fed twice a day, the G7 group exhibited significantly higher TP, GLB, and LZM values than the remaining groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The SOD and AKP values of groups G7-G9 were not significantly different, whereas G6 had the highest values.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Tissues enzyme assays\u003c/h2\u003e \u003cp\u003eAs demonstrated in Table\u0026nbsp;5, there was a significant difference between the gastric, intestinal, and hepatic pepsin and lipase activity between groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Pepsin and lipase activity increased with increasing density regardless of stocking density, with the G5 and G10 groups having the highest values (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), followed by G4 and G9, whereas the G1 and G6 groups had the lowest values. Additionally, no significant differences in gastric, intestinal, and hepatic pepsin and amylase activities were observed between treatments regardless of feeding frequency(\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Tissues histology\u003c/h2\u003e \u003cp\u003eAs illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the vasodilatation of the gastric wall of the G3, G4, G9, and, G10 fish indicated the presence of inflammatory cells in these organisms. Moreover, white vesicles were identified in the cells of fish in the G3, G4, G9, and G10 groups, indicating that the gastric cells in those groups were metamorphosed and became adipogenic. Additionally, gastric tissue sections of other fish groups exhibited varying degrees of inflammation and fat granules except for G7 and G8, which were normal. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows that increasing stocking densities in the fish fed three times a day resulted in lamina propria edemas in the intestinal tissue sections of the G3, G4, and G5 fish, as well as thinner and shorter intestinal villi, some of which were starting to detach. In contrast, the aforementioned symptoms were only identified in the G10 fish that were fed twice a day. Finally, as seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, vacuolation occurred in the G2, G5, G6, G9, and G10 fish, and nuclear deviations were observed in G3 and G4, whereas the hepatocytes in G1, G7, and G8 were normal.\u003c/p\u003e \u003c/div\u003e "},{"header":"4 Discussion","content":"\u003cp\u003eMany factors affect fish growth, including diet, fish size, feeding time, stocking density, and feeding frequency. In this experiment, when the fish were fed three times a day, the BWG, WGR, and SGR of fish stocked at a density of 0.55\u0026nbsp;kg per tank gradually decreased with increasing stocking density and was significantly higher than the other four groups. This suggested that the growth performance of the fish in the 1.10\u0026nbsp;kg and 1.65\u0026nbsp;kg per tank stocking densities was better than that of the other groups with the same feeding frequency. In other words, feeding frequency and stocking density influenced the growth performance of this species, which was consistent with previous studies[\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e]. The poor growth performance observed in the high stocking density was likely due to crowding stress[\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eAdditionally, our findings suggested that feeding three times per day instead of two may have affected satiety levels, thereby resulting in food waste. Several studies have reached different conclusions regarding the effect of stocking density on the growth performance of fish depending on the species. For instance, Rowland et al. (2006) [\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e] reported that stocking density had no significant effects on silver perch. In contrast, high stocking density decreased the final weights of Brook charr according to Vijayan et al. (1990)[\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e], and Boeuf et al. (1989)[\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e] reported that lower densities had positive effects on the growth of Atlantic salmon during smolting. According to the quadratic regression model based on the BWG of the hybrid groupers studied herein, when fed twice a day, the function was y=-0.4872163x4+6.391186x 3-28.19146x2+45.84394x-6.26（R2=1）\u0026nbsp;and we predicted that the optimal stocking density was 2.004\u0026nbsp;kg/m\u003csup\u003e3\u003c/sup\u003e, whereas when the fish were fed three times a day, the function was y=-0.6366255x4+9.53982 x 3-50.46522x2+107.8068x-55.9（R2=1）\u0026nbsp;and the optimal stocking density was 1.343\u0026nbsp;kg/m\u003csup\u003e3\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eBlood can objectively reflect the nutritional metabolism of farmed animals and is an important physiological, pathological, and toxicological indicator. AST and ALT are common indicators of liver cell damage. Specifically, when liver cells are damaged, AST and ALT enzymes will infiltrate into the blood, causing a significant increase in the activity of these enzymes in the blood. Previous studies have linked the occurrence of liver damage with elevated AST and ALT levels [\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e]. In this study, AST levels increased significantly with stocking density, except at a 2.2\u0026nbsp;kg/m\u003csup\u003e3\u003c/sup\u003e. Moreover, ALT decreased significantly with increasing stocking density, particularly at 4.4\u0026nbsp;kg m\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e and 5.5\u0026nbsp;kg m\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e in both feeding frequencies. Our findings suggest that although increases in AST were indicative of some degree of liver damage, lower ALT levels may explain the 100% survival ratio, as it is an important indicator of fish health [\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eSOD can eliminate excess free radicals in organisms [\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e] and modulate the balance between the formation and elimination of free radicals. Therefore, this parameter is an important physiological indicator of antioxidant capacity and non-specific immune function. LZY and AKP are also important indicators of non-specific immunity in the body. AKP is very important for various metabolic processes including phosphorylation and dephosphorylation, as well as nutrient absorption, and its activity can be used as an indicator of fish immune function.\u003c/p\u003e\n\u003cp\u003ePrevious studies have demonstrated that high stocking densities were associated with decreases in SOD activity in some fish [\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e] and shrimps [\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e]. LZY is known to disrupt the structure of the bacterial cell wall, and therefore LZY levels can directly reflect lytic capacity, as well as the impact of pathogens and other environmental factors on fish health. In Senegalese sole, high stocking densities reduced plasma LZY levels[\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e]. A similar pattern was found in juvenile turbot[\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e]. In our study, high stocking densities had negative effects on SOD, AKP, and LZY concentrations, which may explain why high stocking densities often lead to diseases. Nonetheless, other studies have demonstrated that high stocking densities could increase AKP activity[\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e\n\u003cp\u003eTG and CHO are key indicators of blood lipid levels. According to medical research, elevated TG and CHO levels are linked to fatty liver. TP includes GLB and ALB, which are involved in protein metabolism Albumin content generally accounts for more than 50% of total plasma protein content and is known to maintain osmotic pressure balance between blood vessels and tissues, mediate substance binding and transport, protect blood cells and organs, inactivating inflammation, and also acts as an antioxidant and damage repair agent, all of which are closely related to the body\u0026rsquo;s immune function.\u003c/p\u003e\n\u003cp\u003eIn this study, the CHO levels of groupers gradually decreased with increasing stocking density regardless of feeding frequency, meaning that higher stocking densities resulted in more liver cell damage, which would negatively affect fish digestion and fat translation. Moreover, TP and ALB showed a gradual increase, whereas GLB and GLU decreased gradually with increasing stocking density, indicating that the vital activities represented by the above indicators were gradually weakened as the stocking density increased.\u003c/p\u003e\n\u003cp\u003ePrevious studies have shown that the detection and analysis of digestive enzyme activities can directly reflect the physiological status of fish digestion [\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e]. In the present study, increases in stocking density had a positive effect on gastric, intestinal, and hepatic pepsin and lipase activities regardless of feeding frequency. However, these results were not consistent with a previous study in which the lipase, trypsin, and amylase activities of \u003cem\u003eOreochromis niloticus\u003c/em\u003e fingerlings were all depressed in high-density conditions (600 organisms m\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e) [\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e]. This indicated that the fish exhibited better digestive physiology at an appropriate stocking density, which may also explain why the fish had higher growth rates in the corresponding density range. However, the high stocking density in our study may have elicited crowding stress, thereby requiring additional energy expenditure to resist the stress, which in turn required an increased digestive performance. We therefore speculated that increased digestive enzyme activity may be a protective mechanism in high-density conditions; however, this hypothesis must be further explored in future studies.\u003c/p\u003e\n\u003cp\u003eIn this study, feeding frequency and stocking density were found to significantly influence gastric, intestinal, and hepatic tissue histology. When the fish were fed three times a day, even low stocking densities (e.g., 1.1\u0026nbsp;kg m\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e) resulted in pathological signs or symptoms, which was also observed in fish that were fed twice a day and stocked at 2.2\u0026nbsp;kg m\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e and 3.3\u0026nbsp;kg m\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e densities. These results were consistent with those of Refaey et al. (2018)[\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e], who reported that a high stocking density (300 fish m\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e) resulted in intestine and muscle histological changes.\u003c/p\u003e"},{"header":"5 Conclusions","content":"\u003cp\u003eBased on observations of grouper growth performance, digestive enzyme activity, and tissue histology at different stocking densities and feeding frequencies, the optimum stocking density was 1.343\u0026nbsp;kg/m\u003csup\u003e3\u003c/sup\u003e (approximately 10 fish) when the fish were fed three times a day, and 2.004\u0026nbsp;kg/m\u003csup\u003e3\u003c/sup\u003e (approximately 20\u0026ndash;30 fish) when the fish were fed twice a day.\u003c/p\u003e \u003c/div\u003e "},{"header":"Abbreviations","content":"\u003cp\u003eSD: stocking densities; FF: feeding frequencies; WGR: weight gain rate; SGR: specific growth rate; FER: feed efficiency ratio; PER: protein efficiency ratio; HIS: hepatosomatic index ; VSI: viscerosomatic index; IPR: intraperitoneal ratio; BWG: body weight gain; CF: condition factor; H\u0026amp;E; hematoxylin and eosin ; DFI: Daily feed intake; AST: aspartate aminotransferase; ALT; alanine aminotransferase; TGs: TP: total protein; GLB: globin; ALB: albumin; GLU: glucose);triglycerides; CHO: cholesterol; ALB: albumin; LZY: lysozyme; SOD: superoxide dismutase; AKP: alkaline phosphatase; AOAC: association of official analytical chemists; MS222: tricaine methanesulfonate; SD: standard deviation; W\u003csub\u003ei\u003c/sub\u003e : initial weights; W\u003csub\u003ef\u003c/sub\u003e : final weights; WW: wet weight.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e-Ethics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll applicable international, national, and/or institutional guidelines for the care and use of animals were followed by the authors. The use of animal was approved by the Institutional Animal Care and Use Committee of Hainan Tropical Ocean University.\u003c/p\u003e\n\u003cp\u003e-\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003e-Availability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e-Competing interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e-Funding\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study was supported by Basic and Applied basic Research Program of Hainan Province (No. 2019RC246), the Natural Science Foundation of Hainan Province (No. 20163074; No. 20163071), the Major Science and Technology Projects in Hainan Province from Science and Technology Department of Hainan Province government, China (No. ZDKJ2016009), the Hainan Key Laboratory for Conservation and Utilization of Tropical Marine Fishery Resources and Sanya Key Laboratory of Seawater Aquaculture Research (No. L1507).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e-Authors' contributions\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eYan Chen performed the conduct of Experiments, analyzed and interpreted the data regarding growth performance and biochemical analysis of this species, and was a major contributor in writing the manuscript. Huanghai \u0026amp; Jun Ma analyzed and explained the relevant data regarding the tissues enzyme assays and tissues histology of this fish. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e-Acknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThanks are given to the senior and junior students and teachers in College of life science and ecology,Hainan Tropical Ocean University（Sanya, China ）and Hainan Key Laboratory for Conservation and Utilization of Tropical Marine Fishery Resources, for their help in preparing the diets used in this study and assisting in sample collection.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePhillips TA, Summerfelt RC, Clayton RD. Feeding frequency effects on water quality and growth of walleye fingerlings in intensive culture. 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Aquaculture, 2018:S0044848617320744.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable.1 Formulation and proximate composition of the experimental diet.\u003c/p\u003e\n\u003ctable border=\"1\" width=\"0\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"215\"\u003e\n\u003cp\u003eIngredients\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"199\"\u003e\n\u003cp\u003e%\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"215\"\u003e\n\u003cp\u003eFishmeal\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"199\"\u003e\n\u003cp\u003e50.00\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"215\"\u003e\n\u003cp\u003eSoy protein concentrate\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"199\"\u003e\n\u003cp\u003e12.00\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"215\"\u003e\n\u003cp\u003eCasein\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"199\"\u003e\n\u003cp\u003e4.00\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"215\"\u003e\n\u003cp\u003eShrimp meal\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"199\"\u003e\n\u003cp\u003e3.50\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"215\"\u003e\n\u003cp\u003eWheat flour\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"199\"\u003e\n\u003cp\u003e20.5\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"215\"\u003e\n\u003cp\u003eBinding agents\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"199\"\u003e\n\u003cp\u003e2.00\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"215\"\u003e\n\u003cp\u003eSoybean oil\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"199\"\u003e\n\u003cp\u003e1.50\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"215\"\u003e\n\u003cp\u003eFish oil\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"199\"\u003e\n\u003cp\u003e1.50\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"215\"\u003e\n\u003cp\u003eSquid visceral ointment\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"199\"\u003e\n\u003cp\u003e1.50\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"215\"\u003e\n\u003cp\u003eVitamin premix\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"199\"\u003e\n\u003cp\u003e1.00\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"215\"\u003e\n\u003cp\u003eMineral premix\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"199\"\u003e\n\u003cp\u003e1.00\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"215\"\u003e\n\u003cp\u003eCholine chloride\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"199\"\u003e\n\u003cp\u003e0.50\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"215\"\u003e\n\u003cp\u003eMonocalcium Phosphate\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"199\"\u003e\n\u003cp\u003e1.00\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"215\"\u003e\n\u003cp\u003eProximate composition\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"199\"\u003e\n\u003cp\u003e%, dry matter basis\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"215\"\u003e\n\u003cp\u003eCrude protein\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"199\"\u003e\n\u003cp\u003e43.84\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"215\"\u003e\n\u003cp\u003eCrude lipid\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"199\"\u003e\n\u003cp\u003e8.20\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"215\"\u003e\n\u003cp\u003eGross energy (MJ/kg)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"199\"\u003e\n\u003cp\u003e17.56\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eProximate composition values represent means \u0026plusmn; standard deviation of triplicate samples, rounded to the nearest 0.1 g.\u003c/p\u003e\n\u003col\u003e\n\u003cli\u003eObtained from Guangzhou Rifeng Science \u0026amp; Technology Co., LTD (Guangzhou, China).\u003c/li\u003e\n\u003cli\u003eVitamin mixture (mg kg\u003csup\u003e\u0026minus;1\u003c/sup\u003e diet): retinol acetate, 38.0; cholecalciferol, 13.2; a-tocopherol, 210.0; thiamin, 115.0; riboflavin, 380.0 pyridoxine 88.0; pantothenic acid, 368.0; niacin acid, 1030.0 biotin, 10.0; folic acid, 20.0; vitamin B12, 1.3; inositol, 4000.0; ascorbic acid, 500.0 (Ding et al., 2010).\u003c/li\u003e\n\u003cli\u003eMineral mixture (mg kg\u003csup\u003e\u0026minus;1\u003c/sup\u003e diet): MgSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO, 3568.0; NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e\u0026middot;2H\u003csub\u003e2\u003c/sub\u003eO, 25568.0; KCl, 3020.5; KAl (SO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e, 8.3; CoCl2, 28.0; ZnSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO, 353.0; Calactate, 15968.0; CuSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;5H\u003csub\u003e2\u003c/sub\u003eO, 9.0; KI, 7.0; MnSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;4H\u003csub\u003e2\u003c/sub\u003eO, 63.1; Na\u003csub\u003e2\u003c/sub\u003eSeO\u003csub\u003e3\u003c/sub\u003e, 1.5; C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003eFe\u0026middot;5H\u003csub\u003e2\u003c/sub\u003eO, 1533.0; NaCl, 100.0; NaF, 4.0 (Ding et al., 2010).\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable.2 Growth and biometry of juvenile pearl gentian groupers (mean \u0026plusmn; S.D., n=3)\u003c/p\u003e\n\u003ctable border=\"1\" width=\"0\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" width=\"55\"\u003e\n\u003cp\u003eGroup\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"5\" width=\"435\"\u003e\n\u003cp\u003eGrowth performance\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"6\" width=\"455\"\u003e\n\u003cp\u003eBiometric indices\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"92\"\u003e\n\u003cp\u003eW\u003csub\u003ei\u003c/sub\u003e (g)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"88\"\u003e\n\u003cp\u003eW\u003csub\u003ef \u003c/sub\u003e(g)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"82\"\u003e\n\u003cp\u003eBWG(g)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"83\"\u003e\n\u003cp\u003eWGR (%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"92\"\u003e\n\u003cp\u003eSGR(%/d)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003eDFI (%/d)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003eSurvival(%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003eCF(g/cm\u003csup\u003e3\u003c/sup\u003e)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003eHIS (%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"81\"\u003e\n\u003cp\u003eIPR (%)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"55\"\u003e\n\u003cp\u003eG1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"92\"\u003e\n\u003cp\u003e11.03\u0026plusmn;0.09\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"88\"\u003e\n\u003cp\u003e28.88\u0026plusmn;0.003b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"82\"\u003e\n\u003cp\u003e17.85\u0026plusmn;0.11b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"83\"\u003e\n\u003cp\u003e1.62\u0026plusmn;0.02b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"92\"\u003e\n\u003cp\u003e2.29\u0026plusmn;0.02b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e2.65\u0026plusmn;0.01d\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e100.00\u0026plusmn;0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e1.53\u0026plusmn;0.16bcd\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e1.83\u0026plusmn;0.50c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"81\"\u003e\n\u003cp\u003e0.89\u0026plusmn;0.52b\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"55\"\u003e\n\u003cp\u003eG2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"92\"\u003e\n\u003cp\u003e11.10\u0026plusmn;0.05\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"88\"\u003e\n\u003cp\u003e24.89\u0026plusmn;0.02d\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"82\"\u003e\n\u003cp\u003e14.79\u0026plusmn;0.03c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"83\"\u003e\n\u003cp\u003e0.89\u0026plusmn;0.04d\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"92\"\u003e\n\u003cp\u003e2.15\u0026plusmn;0.01cd\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e2.91\u0026plusmn;0.04a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e100.00\u0026plusmn;0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e1.67\u0026plusmn;0.24abc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e1.98\u0026plusmn;0.84bc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"81\"\u003e\n\u003cp\u003e1.27\u0026plusmn;0.61ab\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"55\"\u003e\n\u003cp\u003eG3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"92\"\u003e\n\u003cp\u003e11.22\u0026plusmn;0.18\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"88\"\u003e\n\u003cp\u003e21.14\u0026plusmn;0.06f\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"82\"\u003e\n\u003cp\u003e9.92\u0026plusmn;0.59f\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"83\"\u003e\n\u003cp\u003e0.65\u0026plusmn;0.04e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"92\"\u003e\n\u003cp\u003e1.51\u0026plusmn;0.08g\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e2.23\u0026plusmn;0.01e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e100.00\u0026plusmn;0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e1.56\u0026plusmn;0.23abcd\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e2.72\u0026plusmn;0.83ab\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"81\"\u003e\n\u003cp\u003e1.12\u0026plusmn;0.57ab\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"55\"\u003e\n\u003cp\u003eG4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"92\"\u003e\n\u003cp\u003e11.28\u0026plusmn;0.10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"88\"\u003e\n\u003cp\u003e22.76\u0026plusmn;0.12ef\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"82\"\u003e\n\u003cp\u003e11.48\u0026plusmn;0.12e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"83\"\u003e\n\u003cp\u003e0.85\u0026plusmn;0.03d\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"92\"\u003e\n\u003cp\u003e1.67\u0026plusmn;0.02f\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e2.73\u0026plusmn;0.02c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e100.00\u0026plusmn;0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e1.76\u0026plusmn;0.37ab\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e2.87\u0026plusmn;0.87a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"81\"\u003e\n\u003cp\u003e1.24\u0026plusmn;0.26ab\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"55\"\u003e\n\u003cp\u003eG5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"92\"\u003e\n\u003cp\u003e11.02\u0026plusmn;0.07\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"88\"\u003e\n\u003cp\u003e21.62\u0026plusmn;0.02f\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"82\"\u003e\n\u003cp\u003e10.59\u0026plusmn;0.06f\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"83\"\u003e\n\u003cp\u003e0.73\u0026plusmn;0.01e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"92\"\u003e\n\u003cp\u003e1.60\u0026plusmn;0.01fg\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e2.82\u0026plusmn;0.03b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e100.00\u0026plusmn;0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e1.72\u0026plusmn;0.29abc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e2.13\u0026plusmn;0.64abc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"81\"\u003e\n\u003cp\u003e1.30\u0026plusmn;0.75ab\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"55\"\u003e\n\u003cp\u003eG6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"92\"\u003e\n\u003cp\u003e11.03\u0026plusmn;0.04\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"88\"\u003e\n\u003cp\u003e23.96\u0026plusmn;0.06de\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"82\"\u003e\n\u003cp\u003e13.39\u0026plusmn;0.28d\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"83\"\u003e\n\u003cp\u003e1.27\u0026plusmn;0.06c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"92\"\u003e\n\u003cp\u003e1.95\u0026plusmn;0.06e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e2.74\u0026plusmn;0.01c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e100.00\u0026plusmn;0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e1.38\u0026plusmn;0.16d\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e1.53\u0026plusmn;0.86c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"81\"\u003e\n\u003cp\u003e0.97\u0026plusmn;0.48ab\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"55\"\u003e\n\u003cp\u003eG7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"92\"\u003e\n\u003cp\u003e11.12\u0026plusmn;0.06\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"88\"\u003e\n\u003cp\u003e33.86\u0026plusmn;0.21a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"82\"\u003e\n\u003cp\u003e23.69\u0026plusmn;0.11a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"83\"\u003e\n\u003cp\u003e2.33\u0026plusmn;0.01a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"92\"\u003e\n\u003cp\u003e2.86\u0026plusmn;0.01a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e2.67\u0026plusmn;0.01d\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e100.00\u0026plusmn;0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e1.67\u0026plusmn;0.15abc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e2.95\u0026plusmn;0.62a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"81\"\u003e\n\u003cp\u003e1.28\u0026plusmn;0.52ab\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"55\"\u003e\n\u003cp\u003eG8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"92\"\u003e\n\u003cp\u003e11.17\u0026plusmn;0.07\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"88\"\u003e\n\u003cp\u003e30.18\u0026plusmn;1.72b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"82\"\u003e\n\u003cp\u003e17.63\u0026plusmn;0.38b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"83\"\u003e\n\u003cp\u003e1.29\u0026plusmn;0.02c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"92\"\u003e\n\u003cp\u003e2.26\u0026plusmn;0.04bc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e2.10\u0026plusmn;0.01f\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e100.00\u0026plusmn;0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e1.80\u0026plusmn;0.24a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e2.20\u0026plusmn;0.90abc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"81\"\u003e\n\u003cp\u003e1.41\u0026plusmn;0.67ab\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"55\"\u003e\n\u003cp\u003eG9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"92\"\u003e\n\u003cp\u003e11.32\u0026plusmn;0.07\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"88\"\u003e\n\u003cp\u003e26.79\u0026plusmn;0.08c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"82\"\u003e\n\u003cp\u003e15.47\u0026plusmn;0.08c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"83\"\u003e\n\u003cp\u003e1.26\u0026plusmn;0.01c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"92\"\u003e\n\u003cp\u003e2.05\u0026plusmn;0.01de\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e1.96\u0026plusmn;0.01g\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e100.00\u0026plusmn;0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e1.49\u0026plusmn;0.24cd\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e2.00\u0026plusmn;0.54bc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"81\"\u003e\n\u003cp\u003e1.10\u0026plusmn;0.55ab\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"55\"\u003e\n\u003cp\u003eG10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"92\"\u003e\n\u003cp\u003e11.17\u0026plusmn;0.03\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"88\"\u003e\n\u003cp\u003e26.64\u0026plusmn;0.22c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"82\"\u003e\n\u003cp\u003e15.10\u0026plusmn;0.36c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"83\"\u003e\n\u003cp\u003e1.24\u0026plusmn;0.03c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"92\"\u003e\n\u003cp\u003e1.99\u0026plusmn;0.05e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e1.86\u0026plusmn;0.01h\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e100.00\u0026plusmn;0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e1.58\u0026plusmn;0.15abcd\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e2.80\u0026plusmn;0.73ab\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"81\"\u003e\n\u003cp\u003e1.65\u0026plusmn;0.85a\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"12\" width=\"945\"\u003e\n\u003cp\u003eANOVA (\u003cem\u003eP \u003c/em\u003evalue)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"2\" width=\"148\"\u003e\n\u003cp\u003eBreeding density\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"88\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"82\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"83\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"92\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e0.047\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e0.012\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"81\"\u003e\n\u003cp\u003e0.150\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"2\" width=\"148\"\u003e\n\u003cp\u003eFeeding frequency\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"88\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"82\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"83\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"92\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e0.221\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e0.948\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"81\"\u003e\n\u003cp\u003e0.375\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"2\" width=\"148\"\u003e\n\u003cp\u003eInteractions\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"88\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"82\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"83\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"92\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e0.032\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e0.003\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"81\"\u003e\n\u003cp\u003e0.763\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"55\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd width=\"92\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd width=\"88\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd width=\"82\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd width=\"83\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd width=\"89\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd width=\"3\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd width=\"85\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd width=\"81\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eValues (mean \u0026plusmn; S.D.) with different letters within the same row are significantly different (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05). The absence of letters indicates no significant difference between treatments.\u003c/p\u003e\n\u003cp\u003eANOVA: Two-way analysis of variance \u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 3 Proximate composition of muscle and whole body of juvenile pearl gentian groupers (mean \u0026plusmn; S.D., n=3).\u003c/p\u003e\n\u003ctable border=\"1\" width=\"0\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" width=\"163\"\u003e\n\u003cp\u003eGroup\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"4\" width=\"367\"\u003e\n\u003cp\u003eProximate composition of muscle (% WW\u003csup\u003ea\u003c/sup\u003e)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"4\" width=\"415\"\u003e\n\u003cp\u003eProximate composition of whole body(% WW)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"99\"\u003e\n\u003cp\u003eMoisture\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003eProtein\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003eFat\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003eAsh\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"112\"\u003e\n\u003cp\u003eMoisture\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"107\"\u003e\n\u003cp\u003eProtein\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003eFat\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003eAsh\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"163\"\u003e\n\u003cp\u003eG1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"99\"\u003e\n\u003cp\u003e75.70\u0026plusmn;0.33de\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003e20.05\u0026plusmn;0.37abc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e1.34\u0026plusmn;0.06bcd\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e1.53\u0026plusmn;0.01c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"112\"\u003e\n\u003cp\u003e67.28\u0026plusmn;0.13e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"107\"\u003e\n\u003cp\u003e19.53\u0026plusmn;0.11a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003e6.00\u0026plusmn;0.42ab\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003e6.77\u0026plusmn;0.10a\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"163\"\u003e\n\u003cp\u003eG2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"99\"\u003e\n\u003cp\u003e77.32\u0026plusmn;0.11bcd\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003e18.71\u0026plusmn;0.13cd\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e1.31\u0026plusmn;0.06cde\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e1.54\u0026plusmn;0.01b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"112\"\u003e\n\u003cp\u003e71.64\u0026plusmn;0.07ab\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"107\"\u003e\n\u003cp\u003e17.78\u0026plusmn;0.04b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003e5.06\u0026plusmn;0.46ab\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003e5.32\u0026plusmn;0.01d\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"163\"\u003e\n\u003cp\u003eG3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"99\"\u003e\n\u003cp\u003e78.69\u0026plusmn;0.21ab\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003e17.20\u0026plusmn;0.20e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e1.16\u0026plusmn;0.07ef\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e1.46\u0026plusmn;0.01e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"112\"\u003e\n\u003cp\u003e71.21\u0026plusmn;0.12b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"107\"\u003e\n\u003cp\u003e18.12\u0026plusmn;0.07b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003e4.05\u0026plusmn;0.06b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003e5.28\u0026plusmn;0.01d\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"163\"\u003e\n\u003cp\u003eG4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"99\"\u003e\n\u003cp\u003e78.47\u0026plusmn;0.26ab\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003e17.65\u0026plusmn;0.23de\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e1.43\u0026plusmn;0.03bc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e1.46\u0026plusmn;0.01e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"112\"\u003e\n\u003cp\u003e71.73\u0026plusmn;0.19ab\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"107\"\u003e\n\u003cp\u003e18.21\u0026plusmn;0.25b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003e4.23\u0026plusmn;0.06ab\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003e4.84\u0026plusmn;0.01f\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"163\"\u003e\n\u003cp\u003eG5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"99\"\u003e\n\u003cp\u003e79.85\u0026plusmn;0.39a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003e16.36\u0026plusmn;0.29e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e1.39\u0026plusmn;0.05bcd\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e1.33\u0026plusmn;0.01g\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"112\"\u003e\n\u003cp\u003e71.24\u0026plusmn;0.09b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"107\"\u003e\n\u003cp\u003e18.23\u0026plusmn;0.25b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003e4.77\u0026plusmn;0.05ab\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003e5.08\u0026plusmn;0.03e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"163\"\u003e\n\u003cp\u003eG6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"99\"\u003e\n\u003cp\u003e77.48\u0026plusmn;1.54bc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003e19.06\u0026plusmn;1.31bc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e1.03\u0026plusmn;0.07f\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e1.49\u0026plusmn;0.01d\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"112\"\u003e\n\u003cp\u003e66.94\u0026plusmn;0.23e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"107\"\u003e\n\u003cp\u003e20.65\u0026plusmn;0.30a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003e7.49\u0026plusmn;1.87ab\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003e6.38\u0026plusmn;0.03b\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"163\"\u003e\n\u003cp\u003eG7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"99\"\u003e\n\u003cp\u003e76.51\u0026plusmn;0.23cde\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003e20.28\u0026plusmn;0.32ab\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e1.74\u0026plusmn;0.03a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e1.44\u0026plusmn;0.01f\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"112\"\u003e\n\u003cp\u003e68.00\u0026plusmn;0.20d\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"107\"\u003e\n\u003cp\u003e20.61\u0026plusmn;0.16a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003e5.41\u0026plusmn;0.07ab\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003e5.49\u0026plusmn;0.05c\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"163\"\u003e\n\u003cp\u003eG8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"99\"\u003e\n\u003cp\u003e75.18\u0026plusmn;0.21e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003e21.05\u0026plusmn;0.10a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e1.48\u0026plusmn;0.06b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e1.54\u0026plusmn;0.01b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"112\"\u003e\n\u003cp\u003e68.07\u0026plusmn;0.21d\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"107\"\u003e\n\u003cp\u003e20.93\u0026plusmn;0.19a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003e8.58\u0026plusmn;3.78a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003e5.32\u0026plusmn;0.03d\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"163\"\u003e\n\u003cp\u003eG9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"99\"\u003e\n\u003cp\u003e76.22\u0026plusmn;0.17cde\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003e20.37\u0026plusmn;0.13ab\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e1.38\u0026plusmn;0.02bcd\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e1.57\u0026plusmn;0.01a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"112\"\u003e\n\u003cp\u003e71.78\u0026plusmn;0.19a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"107\"\u003e\n\u003cp\u003e18.07\u0026plusmn;0.47b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003e4.37\u0026plusmn;0.07ab\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003e4.98\u0026plusmn;0.01e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"163\"\u003e\n\u003cp\u003eG10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"99\"\u003e\n\u003cp\u003e77.25\u0026plusmn;0.18bcd\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003e19.68\u0026plusmn;0.16abc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e1.24\u0026plusmn;0.04de\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e1.46\u0026plusmn;0.01e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"112\"\u003e\n\u003cp\u003e70.56\u0026plusmn;0.22c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"107\"\u003e\n\u003cp\u003e18.12\u0026plusmn;0.12b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003e4.36\u0026plusmn;0.06ab\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003e5.25\u0026plusmn;0.01d\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"9\" width=\"945\"\u003e\n\u003cp\u003eANOVA(\u003cem\u003eP\u003c/em\u003e value)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"163\"\u003e\n\u003cp\u003eBreeding density\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"99\"\u003e\n\u003cp\u003e0.005\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003e0.012\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"112\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"107\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003e0.295\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"163\"\u003e\n\u003cp\u003eFeeding frequency\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"99\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e0.139\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"112\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"107\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003e0.157\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003e0.250\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"163\"\u003e\n\u003cp\u003eInteractions\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"99\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"94\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"87\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"112\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"107\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003e0.377\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"98\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eValues (mean \u0026plusmn; S.D.) with different letters within the same row are significantly different (P\u0026lt;0.05). The absence of letters indicates no significant difference between treatments.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ea\u003c/sup\u003e WW: wet weight.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable.4 Serum biochemistry (mean \u0026plusmn; S.D., n=3).\u003c/p\u003e\n\u003ctable border=\"1\" width=\"0\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd rowspan=\"2\" width=\"76\"\u003e\n\u003cp\u003e\u003cu\u003eGroup\u003c/u\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"20\" width=\"936\"\u003e\n\u003cp\u003eItems\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"2\" width=\"95\"\u003e\n\u003cp\u003eAST\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003eALT\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"81\"\u003e\n\u003cp\u003eTP\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"90\"\u003e\n\u003cp\u003eGLB\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"84\"\u003e\n\u003cp\u003eALB\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"75\"\u003e\n\u003cp\u003eTG\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"75\"\u003e\n\u003cp\u003eCHO\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"85\"\u003e\n\u003cp\u003eGLU\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"86\"\u003e\n\u003cp\u003eLZM\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"76\"\u003e\n\u003cp\u003eSOD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003eAKP\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eG1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"95\"\u003e\n\u003cp\u003e17.33\u0026plusmn;0.58h\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003e269.00\u0026plusmn;8.39a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"81\"\u003e\n\u003cp\u003e30.50\u0026plusmn;0.06e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"90\"\u003e\n\u003cp\u003e21.03\u0026plusmn;0.12bc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"84\"\u003e\n\u003cp\u003e9.47\u0026plusmn;0.07f\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"75\"\u003e\n\u003cp\u003e0.99\u0026plusmn;0.01fg\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"75\"\u003e\n\u003cp\u003e1.65\u0026plusmn;0.01h\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"85\"\u003e\n\u003cp\u003e22.60\u0026plusmn;0.08b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"86\"\u003e\n\u003cp\u003e91.41\u0026plusmn;4.82e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"76\"\u003e\n\u003cp\u003e4.46\u0026plusmn;0.09a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e14.53\u0026plusmn;0.13a\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eG2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"95\"\u003e\n\u003cp\u003e52.33\u0026plusmn;0.58d\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003e152.00\u0026plusmn;3.46b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"81\"\u003e\n\u003cp\u003e26.97\u0026plusmn;0.09h\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"90\"\u003e\n\u003cp\u003e17.27\u0026plusmn;0.13f\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"84\"\u003e\n\u003cp\u003e9.70\u0026plusmn;0.06ef\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"75\"\u003e\n\u003cp\u003e1.31\u0026plusmn;0.02c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"75\"\u003e\n\u003cp\u003e1.87\u0026plusmn;0.04g\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"85\"\u003e\n\u003cp\u003e20.45\u0026plusmn;0.16c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"86\"\u003e\n\u003cp\u003e76.67\u0026plusmn;5.09f\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"76\"\u003e\n\u003cp\u003e4.73\u0026plusmn;0.14c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e13.80 \u0026plusmn;0.03b\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eG3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"95\"\u003e\n\u003cp\u003e33.00\u0026plusmn;1.00f\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003e138.33\u0026plusmn;6.57b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"81\"\u003e\n\u003cp\u003e29.37\u0026plusmn;0.15fg\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"90\"\u003e\n\u003cp\u003e19.37\u0026plusmn;0.09d\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"84\"\u003e\n\u003cp\u003e10.00\u0026plusmn;0.10e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"75\"\u003e\n\u003cp\u003e0.90\u0026plusmn;0.03h\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"75\"\u003e\n\u003cp\u003e2.38\u0026plusmn;0.02f\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"85\"\u003e\n\u003cp\u003e9.73\u0026plusmn;0.04e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"86\"\u003e\n\u003cp\u003e58.89\u0026plusmn;4.84gh\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"76\"\u003e\n\u003cp\u003e4.51\u0026plusmn;0.06a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e13.33\u0026plusmn;0.10c\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eG4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"95\"\u003e\n\u003cp\u003e53.33\u0026plusmn;0.58d\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003e52.67\u0026plusmn;1.76c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"81\"\u003e\n\u003cp\u003e36.87\u0026plusmn;0.17a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"90\"\u003e\n\u003cp\u003e21.53\u0026plusmn;0.07a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"84\"\u003e\n\u003cp\u003e15.33\u0026plusmn;0.15a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"75\"\u003e\n\u003cp\u003e1.03\u0026plusmn;0.03ef\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"75\"\u003e\n\u003cp\u003e2.76\u0026plusmn;0.01d\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"85\"\u003e\n\u003cp\u003e8.57\u0026plusmn;0.04e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"86\"\u003e\n\u003cp\u003e53.33\u0026plusmn;3.33h\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"76\"\u003e\n\u003cp\u003e4.35\u0026plusmn;0.03a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e12.37 \u0026plusmn;0.08d\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eG5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"95\"\u003e\n\u003cp\u003e60.00\u0026plusmn;0.00c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003e47.67\u0026plusmn;2.33c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"81\"\u003e\n\u003cp\u003e31.30\u0026plusmn;0.10d\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"90\"\u003e\n\u003cp\u003e17.17\u0026plusmn;0.07f\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"84\"\u003e\n\u003cp\u003e14.13\u0026plusmn;0.15b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"75\"\u003e\n\u003cp\u003e0.78\u0026plusmn;0.03i\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"75\"\u003e\n\u003cp\u003e2.63\u0026plusmn;0.01e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"85\"\u003e\n\u003cp\u003e12.70\u0026plusmn;0.10d\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"86\"\u003e\n\u003cp\u003e66.67\u0026plusmn;0.00fg\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"76\"\u003e\n\u003cp\u003e4.01\u0026plusmn;0.13b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e10.54\u0026plusmn;0.09f\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eG6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"95\"\u003e\n\u003cp\u003e21.00\u0026plusmn;1.00g\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003e267.67\u0026plusmn;2.03a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"81\"\u003e\n\u003cp\u003e29.07\u0026plusmn;0.03g\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"90\"\u003e\n\u003cp\u003e20.67\u0026plusmn;0.15c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"84\"\u003e\n\u003cp\u003e8.40\u0026plusmn;0.15g\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"75\"\u003e\n\u003cp\u003e1.53\u0026plusmn;0.02b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"75\"\u003e\n\u003cp\u003e1.44\u0026plusmn;0.03i\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"85\"\u003e\n\u003cp\u003e2.05\u0026plusmn;0.03f\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"86\"\u003e\n\u003cp\u003e166.67\u0026plusmn;0.00c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"76\"\u003e\n\u003cp\u003e4.00\u0026plusmn;0.11b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e11.50 \u0026plusmn;0.06e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eG7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"95\"\u003e\n\u003cp\u003e39.00\u0026plusmn;1.73e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003e258.33\u0026plusmn;9.94a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"81\"\u003e\n\u003cp\u003e33.20\u0026plusmn;0.15b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"90\"\u003e\n\u003cp\u003e21.97\u0026plusmn;0.07a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"84\"\u003e\n\u003cp\u003e8.23\u0026plusmn;0.19g\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"75\"\u003e\n\u003cp\u003e0.93\u0026plusmn;0.03gh\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"75\"\u003e\n\u003cp\u003e3.56\u0026plusmn;0.03b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"85\"\u003e\n\u003cp\u003e13.63\u0026plusmn;0.19d\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"86\"\u003e\n\u003cp\u003e285.55\u0026plusmn;5.37a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"76\"\u003e\n\u003cp\u003e3.77\u0026plusmn;0.06bc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e7.38 \u0026plusmn;0.08g\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eG8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"95\"\u003e\n\u003cp\u003e18.00\u0026plusmn;1.00h\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003e108.67\u0026plusmn;5.04b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"81\"\u003e\n\u003cp\u003e29.73\u0026plusmn;0.23f\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"90\"\u003e\n\u003cp\u003e21.43\u0026plusmn;0.35ab\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"84\"\u003e\n\u003cp\u003e8.30\u0026plusmn;0.15g\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"75\"\u003e\n\u003cp\u003e1.65\u0026plusmn;0.01a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"75\"\u003e\n\u003cp\u003e3.37\u0026plusmn;0.01c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"85\"\u003e\n\u003cp\u003e38.37\u0026plusmn;1.39a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"86\"\u003e\n\u003cp\u003e201.11\u0026plusmn;1.92b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"76\"\u003e\n\u003cp\u003e3.76\u0026plusmn;0.02bc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e7.15 \u0026plusmn;0.05g\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eG9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"95\"\u003e\n\u003cp\u003e69.67\u0026plusmn;1.15b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003e36.67\u0026plusmn;0.33c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"81\"\u003e\n\u003cp\u003e30.70\u0026plusmn;0.26e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"90\"\u003e\n\u003cp\u003e18.43\u0026plusmn;0.18e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"84\"\u003e\n\u003cp\u003e12.27\u0026plusmn;0.09d\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"75\"\u003e\n\u003cp\u003e1.08\u0026plusmn;0.02e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"75\"\u003e\n\u003cp\u003e2.78\u0026plusmn;0.01d\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"85\"\u003e\n\u003cp\u003e22.87\u0026plusmn;0.08b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"86\"\u003e\n\u003cp\u003e277.78\u0026plusmn;9.62a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"76\"\u003e\n\u003cp\u003e3.57\u0026plusmn;0.02c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e7.17 \u0026plusmn;0.03g\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\n\u003cp\u003eG10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"95\"\u003e\n\u003cp\u003e77.33\u0026plusmn;0.58a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"84\"\u003e\n\u003cp\u003e43.67\u0026plusmn;1.33c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"81\"\u003e\n\u003cp\u003e32.10\u0026plusmn;0.10c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"90\"\u003e\n\u003cp\u003e18.83\u0026plusmn;0.09e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"84\"\u003e\n\u003cp\u003e13.27\u0026plusmn;0.12c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"75\"\u003e\n\u003cp\u003e1.19\u0026plusmn;0.03d\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"75\"\u003e\n\u003cp\u003e4.10\u0026plusmn;0.07a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"85\"\u003e\n\u003cp\u003e22.55\u0026plusmn;0.24b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"86\"\u003e\n\u003cp\u003e108.33\u0026plusmn;8.34d\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"76\"\u003e\n\u003cp\u003e1.56\u0026plusmn;0.04d\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\n\u003cp\u003e7.58 \u0026plusmn;0.02f\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"21\" width=\"1011\"\u003e\n\u003cp\u003eANOVA(\u003cem\u003eP\u003c/em\u003e value)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"2\" width=\"93\"\u003e\n\u003cp\u003eBreeding density\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"78\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"85\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"80\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"75\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"85\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"66\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"76\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"75\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"85\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"76\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"136\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"2\" width=\"93\"\u003e\n\u003cp\u003eFeeding frequency\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"78\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"85\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"80\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"75\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"85\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"66\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"76\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"75\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"85\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"76\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"136\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"2\" width=\"93\"\u003e\n\u003cp\u003eInteractions\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"78\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"85\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"80\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"75\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"85\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"66\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"76\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"75\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"85\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"76\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd colspan=\"2\" width=\"136\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"76\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd width=\"17\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd width=\"78\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd width=\"84\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd width=\"1\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd width=\"80\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd width=\"75\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd width=\"15\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd width=\"70\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd width=\"14\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd width=\"52\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd width=\"23\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd width=\"53\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd width=\"23\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd width=\"53\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd width=\"32\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd width=\"53\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd width=\"33\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd width=\"43\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd width=\"33\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd width=\"104\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eValues (mean \u0026plusmn; S.D.) with different letters within the same row are significantly different (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05). The absence of letters indicates no significant difference between treatments.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ea \u003c/sup\u003eWW: wet weight.\u003c/p\u003e\n\u003cp\u003eAST: Aspartate aminotransferase; ALT: Alanine transaminase; TP: Total protein; GLB: Globulin; ALB: Albumin; TG: Triglyceride; CHO: Cholesterol; GLU: Glucose; LZY: Serum lysozyme; SOD: Superoxide dismutase; AKP: Alkaline phosphatase\u003c/p\u003e\n\u003cp\u003eTable.5 Digestive enzyme activities (mean \u0026plusmn; S.D., n=3).\u003c/p\u003e\n\u003ctable border=\"1\" width=\"0\"\u003e\n\u003ctbody\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 83px;\" rowspan=\"2\" width=\"132\"\u003e\n\u003cp\u003e\u003cu\u003eGroup\u003c/u\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" colspan=\"3\" width=\"283\"\u003e\n\u003cp\u003eGastric\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" colspan=\"4\" width=\"238\"\u003e\n\u003cp\u003eIntestinal\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" colspan=\"4\" width=\"292\"\u003e\n\u003cp\u003eHepatic\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 48px;\"\u003e\n\u003ctd style=\"height: 48px;\" width=\"97\"\u003e\n\u003cp\u003ePepsin (U/mg prot)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 48px;\" width=\"88\"\u003e\n\u003cp\u003eAmylase (U/mg prot)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 48px;\" width=\"98\"\u003e\n\u003cp\u003eLipase (U/mg prot)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 48px;\" width=\"91\"\u003e\n\u003cp\u003ePepsin (U/mg prot)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 48px;\" width=\"81\"\u003e\n\u003cp\u003eAmylase (U/mg prot)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 48px;\" colspan=\"3\" width=\"95\"\u003e\n\u003cp\u003eLipase (U/mg prot)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 48px;\" width=\"91\"\u003e\n\u003cp\u003ePepsin (U/mg prot)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 48px;\" width=\"81\"\u003e\n\u003cp\u003eAmylase (U/mg prot)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 48px;\" width=\"91\"\u003e\n\u003cp\u003eLipase (U/mg prot)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" width=\"132\"\u003e\n\u003cp\u003eG1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"97\"\u003e\n\u003cp\u003e17.23\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"88\"\u003e\n\u003cp\u003e0.68\u0026plusmn;0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"98\"\u003e\n\u003cp\u003e18.08\u0026plusmn;0.12f\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"91\"\u003e\n\u003cp\u003e1.54\u0026plusmn;0.01d\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"81\"\u003e\n\u003cp\u003e0.26\u0026plusmn;0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" colspan=\"3\" width=\"95\"\u003e\n\u003cp\u003e30.61\u0026plusmn;0.13ef\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"91\"\u003e\n\u003cp\u003e4.66\u0026plusmn;0.01d\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"81\"\u003e\n\u003cp\u003e0.10\u0026plusmn;0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"91\"\u003e\n\u003cp\u003e2.23\u0026plusmn;0.04d\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" width=\"132\"\u003e\n\u003cp\u003eG2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"97\"\u003e\n\u003cp\u003e18.48\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"88\"\u003e\n\u003cp\u003e0.71\u0026plusmn;\u0026plusmn;0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"98\"\u003e\n\u003cp\u003e19.11\u0026plusmn;0.11e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"91\"\u003e\n\u003cp\u003e1.62\u0026plusmn;0.01c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"81\"\u003e\n\u003cp\u003e0.25\u0026plusmn;0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" colspan=\"3\" width=\"95\"\u003e\n\u003cp\u003e31.76\u0026plusmn;0.11d\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"91\"\u003e\n\u003cp\u003e5.03\u0026plusmn;0.01cd\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"81\"\u003e\n\u003cp\u003e0.11\u0026plusmn;0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"91\"\u003e\n\u003cp\u003e2.56\u0026plusmn;0.04cd\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" width=\"132\"\u003e\n\u003cp\u003eG3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"97\"\u003e\n\u003cp\u003e20.67\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"88\"\u003e\n\u003cp\u003e0.77\u0026plusmn;0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"98\"\u003e\n\u003cp\u003e20.0\u0026plusmn;0.14ad\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"91\"\u003e\n\u003cp\u003e1.68\u0026plusmn;0.01bc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"81\"\u003e\n\u003cp\u003e0.24\u0026plusmn;0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" colspan=\"3\" width=\"95\"\u003e\n\u003cp\u003e32.33\u0026plusmn;0.16c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"91\"\u003e\n\u003cp\u003e5.37\u0026plusmn;0.05bc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"81\"\u003e\n\u003cp\u003e0.12\u0026plusmn;0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"91\"\u003e\n\u003cp\u003e2.65\u0026plusmn;0.08c\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" width=\"132\"\u003e\n\u003cp\u003eG4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"97\"\u003e\n\u003cp\u003e22.32\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"88\"\u003e\n\u003cp\u003e0.80\u0026plusmn;0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"98\"\u003e\n\u003cp\u003e20.71\u0026plusmn;0.21ab\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"91\"\u003e\n\u003cp\u003e1.76\u0026plusmn;0.02ab\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"81\"\u003e\n\u003cp\u003e0.23\u0026plusmn;0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" colspan=\"3\" width=\"95\"\u003e\n\u003cp\u003e33.07\u0026plusmn;0.20b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"91\"\u003e\n\u003cp\u003e5.44\u0026plusmn;0.00bc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"81\"\u003e\n\u003cp\u003e0.12\u0026plusmn;0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"91\"\u003e\n\u003cp\u003e2.90\u0026plusmn;0.03ab\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" width=\"132\"\u003e\n\u003cp\u003eG5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"97\"\u003e\n\u003cp\u003e24.21\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"88\"\u003e\n\u003cp\u003e0.84\u0026plusmn;0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"98\"\u003e\n\u003cp\u003e21.18\u0026plusmn;0.25a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"91\"\u003e\n\u003cp\u003e1.78\u0026plusmn;0.01ab\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"81\"\u003e\n\u003cp\u003e0.22\u0026plusmn;0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" colspan=\"3\" width=\"95\"\u003e\n\u003cp\u003e34.80\u0026plusmn;0.12a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"91\"\u003e\n\u003cp\u003e5.94\u0026plusmn;0.01b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"81\"\u003e\n\u003cp\u003e0.13\u0026plusmn;0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"91\"\u003e\n\u003cp\u003e2.97\u0026plusmn;0.05a\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" width=\"132\"\u003e\n\u003cp\u003eG6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"97\"\u003e\n\u003cp\u003e17.38\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"88\"\u003e\n\u003cp\u003e0.70\u0026plusmn;0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"98\"\u003e\n\u003cp\u003e17.63\u0026plusmn;0.15f\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"91\"\u003e\n\u003cp\u003e1.57\u0026plusmn;0.02cd\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"81\"\u003e\n\u003cp\u003e0.28\u0026plusmn;0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" colspan=\"3\" width=\"95\"\u003e\n\u003cp\u003e30.33\u0026plusmn;0.21f\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"91\"\u003e\n\u003cp\u003e4.87\u0026plusmn;0.01dc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"81\"\u003e\n\u003cp\u003e0.10\u0026plusmn;0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"91\"\u003e\n\u003cp\u003e2.12\u0026plusmn;0.04e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" width=\"132\"\u003e\n\u003cp\u003eG7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"97\"\u003e\n\u003cp\u003e19.48\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"88\"\u003e\n\u003cp\u003e0.74\u0026plusmn;0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"98\"\u003e\n\u003cp\u003e18.65\u0026plusmn;0.20e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"91\"\u003e\n\u003cp\u003e1.63\u0026plusmn;0.01c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"81\"\u003e\n\u003cp\u003e0.23\u0026plusmn;0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" colspan=\"3\" width=\"95\"\u003e\n\u003cp\u003e30.85\u0026plusmn;0.10e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"91\"\u003e\n\u003cp\u003e5.19\u0026plusmn;0.01c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"81\"\u003e\n\u003cp\u003e0.11\u0026plusmn;0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"91\"\u003e\n\u003cp\u003e2.24\u0026plusmn;0.06d\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" width=\"132\"\u003e\n\u003cp\u003eG8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"97\"\u003e\n\u003cp\u003e22.57bc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"88\"\u003e\n\u003cp\u003e0.80\u0026plusmn;0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"98\"\u003e\n\u003cp\u003e19.65\u0026plusmn;0.19d\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"91\"\u003e\n\u003cp\u003e1.72\u0026plusmn;0.01b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"81\"\u003e\n\u003cp\u003e0.22\u0026plusmn;0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" colspan=\"3\" width=\"95\"\u003e\n\u003cp\u003e31.42\u0026plusmn;0.09d\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"91\"\u003e\n\u003cp\u003e5.61\u0026plusmn;0.01cd\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"81\"\u003e\n\u003cp\u003e0.11\u0026plusmn;0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"91\"\u003e\n\u003cp\u003e2.68\u0026plusmn;0.06c\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" width=\"132\"\u003e\n\u003cp\u003eG9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"97\"\u003e\n\u003cp\u003e23.62\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"88\"\u003e\n\u003cp\u003e0.83\u0026plusmn;0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"98\"\u003e\n\u003cp\u003e19.84\u0026plusmn;0.16d\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"91\"\u003e\n\u003cp\u003e1.77\u0026plusmn;0.01ab\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"81\"\u003e\n\u003cp\u003e0.22\u0026plusmn;0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" colspan=\"3\" width=\"95\"\u003e\n\u003cp\u003e32.23\u0026plusmn;0.12c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"91\"\u003e\n\u003cp\u003e5.88\u0026plusmn;0.01bc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"81\"\u003e\n\u003cp\u003e0.12\u0026plusmn;0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"91\"\u003e\n\u003cp\u003e2.79\u0026plusmn;0.06b\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" width=\"132\"\u003e\n\u003cp\u003eG10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"97\"\u003e\n\u003cp\u003e25.03\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"88\"\u003e\n\u003cp\u003e0.86\u0026plusmn;0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"98\"\u003e\n\u003cp\u003e20.61\u0026plusmn;0.25bc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"91\"\u003e\n\u003cp\u003e1.80\u0026plusmn;0.02a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"81\"\u003e\n\u003cp\u003e0.21\u0026plusmn;0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" colspan=\"3\" width=\"95\"\u003e\n\u003cp\u003e33.21\u0026plusmn;0.12b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"91\"\u003e\n\u003cp\u003e6.24\u0026plusmn;0.00a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"81\"\u003e\n\u003cp\u003e0.14\u0026plusmn;0.00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"91\"\u003e\n\u003cp\u003e2.88\u0026plusmn;0.04ab\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" colspan=\"12\" width=\"945\"\u003e\n\u003cp\u003eANOVA(Pvalue)\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" width=\"132\"\u003e\n\u003cp\u003eBreeding\u0026nbsp;density\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"97\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"88\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"98\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"91\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" colspan=\"2\" width=\"96\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" colspan=\"2\" width=\"80\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"91\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"81\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"91\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" width=\"132\"\u003e\n\u003cp\u003eFeeding frequency\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"97\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"88\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"98\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"91\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" colspan=\"2\" width=\"96\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" colspan=\"2\" width=\"80\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"91\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"81\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"91\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 35px;\"\u003e\n\u003ctd style=\"height: 35px;\" width=\"132\"\u003e\n\u003cp\u003eInteractions\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"97\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"88\"\u003e\n\u003cp\u003e0.136\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"98\"\u003e\n\u003cp\u003e0.734\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"91\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" colspan=\"2\" width=\"96\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" colspan=\"2\" width=\"80\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"91\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"81\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"height: 35px;\" width=\"91\"\u003e\n\u003cp\u003e\u0026lt; 0.001\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 13px;\"\u003e\n\u003ctd style=\"height: 13px;\" width=\"132\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"height: 13px;\" width=\"97\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"height: 13px;\" width=\"88\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"height: 13px;\" width=\"98\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"height: 13px;\" width=\"91\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"height: 13px;\" width=\"81\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"height: 13px;\" width=\"15\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"height: 13px;\" width=\"50\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"height: 13px;\" width=\"29\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"height: 13px;\" width=\"91\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"height: 13px;\" width=\"81\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"height: 13px;\" width=\"91\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eValues (mean \u0026plusmn; S.D.) with different letters within the same row are significantly different (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05). The absence of letters indicates no significant difference between treatments.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ea \u003c/sup\u003eWW: wet weight.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"hybrid grouper, stocking density, feeding frequency, growth performance, digestive enzyme activity, tissue histology","lastPublishedDoi":"10.21203/rs.3.rs-103762/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-103762/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA 6-week 5×2 factorial study was conducted to examine the effects of stocking density and feeding frequency on growth performance, body composition, digestive enzyme activity, and tissue histology of juvenile hybrid groupers raised in an indoor circulating water system. Triplicate groups of fish were reared in tanks following a factorial design consisting of ten treatments including 5 stocking densities (SD) [1.1 kg m\u003csup\u003e−3\u003c/sup\u003e(0.55 kg fish per tank, SD1.1), 2.2 kg m\u003csup\u003e-3\u003c/sup\u003e(1.1 kg fish per tank, SD2.2), 3.3 kg m\u003csup\u003e−3\u003c/sup\u003e(1.65 kg fish per tank, SD3.3), 4.4 kg m\u003csup\u003e−3\u003c/sup\u003e(2.2 kg fish per tank, SD4.4) and 5.5 kg m\u003csup\u003e−3\u003c/sup\u003e(2.75 kg fish per tank, SD 5.5)] and 2 feeding frequencies (FF) [3 meals a day (FF3) and 2 meals a day (FF2)]. The resulting 10 treatments were G1(SD1.1,FF3), G2(SD2.2,FF3), G3(SD3.3,FF3), G4(SD4.4,FF3), G5(SD5.5,FF3), G6(SD1.1,FF2), G7(SD2.2, FF2), G8(SD3.3, FF2), G9(SD4.4, FF2) andG10 (SD5.5, FF2). Feed consumption and temperature were recorded throughout the experiment. After 6 weeks, the results indicated that the weight gain rate (WGR) and specific growth rate (SGR) of fish in the G7 group were significantly higher than those of other groups (\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05), followed by G1, with G3 being the lowest. Weight gain and specific growth rates were generally higher in fish fed twice a day than those fed three times a day. The variations in protein content between groups were consistent with the muscle protein content trends. Feeding frequency and stocking density had significant effects on serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), triglycerides (TGs), and cholesterol (CHO) (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05). Regarding immune function, grouper albumin (ALB), serum lysozyme (LZY), superoxide dismutase (SOD), and alkaline phosphatase (AKP) were significantly affected by stocking density and feeding frequency (\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05). Pepsin and lipase activities in the stomach, intestine, and liver were also affected. The histological structure of the stomach, liver, and intestine in G1, G2, G7, and G8 fish was relatively normal, whereas those of the remaining groups exhibited varying degrees of damage. Overall, the optimum stocking densities were \u0026nbsp;1.343 kg/m\u003csup\u003e3\u003c/sup\u003e (approximately 10 fish) and 2.004 kg/m\u003csup\u003e3\u003c/sup\u003e (approximately 20-30 fish) when the fish were fed 3 and 2 times per day, respectively.\u003c/p\u003e","manuscriptTitle":"Effects of\u0026nbsp;Stocking\u0026nbsp;Density and FeedingfFrequency on the Growth Performance,\u0026nbsp;Digestive Enzyme Activity, and Tissues Histology of\u0026nbsp;Hybrid Grouper (Epinephelus Fuscoguttatus♀×E. Lanceolatus♂)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-11-12 16:46:25","doi":"10.21203/rs.3.rs-103762/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9562e3b2-7cc2-4853-88ae-b6c3fc2c4913","owner":[],"postedDate":"November 12th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":1057368,"name":"Medical Genetics"},{"id":1057369,"name":"Molecular Genetics"},{"id":1057370,"name":"Population Genetics"}],"tags":[],"updatedAt":"2020-12-20T12:43:58+00:00","versionOfRecord":[],"versionCreatedAt":"2020-11-12 16:46:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-103762","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-103762","identity":"rs-103762","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}