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  (  cy969398hn@126.com )
Hainan Tropical Ocean University https://orcid.org/0000-0002-9922-0489
Hai Huang 
Hainan Tropical Ocean University
Jun Ma 
Hainan Tropical Ocean University
Wenkan Liu 
Hainan Tropical Ocean University
Honggan Zhong 
Hainan Tropical Ocean University
Jiechao Zhang 
Hainan Tropical Ocean University
Jiaohao Zhong 
Hainan Tropical Ocean University
Feng Ye 
Hainan Tropical Ocean University
Chengcai Li 
Hainan Tropical Ocean University
Xiaomei Wang 
Hainan Tropical Ocean University
Chenxi Xu 
Hainan Tropical Ocean University
Jiajie Zhu 
Hainan Tropical Ocean University

Research article

Keywords: hybrid grouper, stocking density, feeding frequency, growth performance, digestive enzyme activity, tissue histology

DOI: https://doi.org/10.21203/rs.3.rs-103762/v1

License:   This work is licensed under a Creative Commons Attribution 4.0 International License.   Read Full License

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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 sh 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 sh per tank, SD1.1), 2.2 kg m-3(1.1 kg
sh per tank, SD2.2), 3.3 kg m−3(1.65 kg sh per tank, SD3.3), 4.4 kg m−3(2.2 kg sh per tank, SD4.4) and 5.5 kg m−3(2.75 kg sh 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 speci c growth rate (SGR) of sh in the G7
group were signi cantly higher than those of other groups (P< 0.05), followed by G1, with G3 being the lowest. Weight gain and speci c growth rates were
generally higher in sh 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 signi cant 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 signi cantly 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 sh was relatively normal,
whereas those of the remaining groups exhibited varying degrees of damage. Overall, the optimum stocking densities were  1.343 kg/m3 (approximately 10
sh) and 2.004 kg/m3 (approximately 20-30 sh) when the sh were fed 3 and 2 times per day, respectively.

Background
The balance between rapid sh growth, feed utilization e ciency, feeding strategy, and intensive sh-farming practices is a key point in sh aquaculture [1, 2].
Feeding time and frequency have been reported to affect feed intake and growth performance in different sh species including the Australian snapper Pagrus
auratus [3], pikeperch Sander lucioperca [4], African cat sh Clariasgariepinus, Burchell 1822 [5], ounder Platichthys esusluscus [6] and rock bream
Oplegnathusfasciatus [7]. Water quality, management procedures, stocking density, feeding frequency, and nutritional conditions are all directly associated
with sh stress [8, 9, 10].

Feeding frequency is a well-known regulator of growth performance, feed intake, and chemical composition of sh, and therefore optimizing this factor can
substantially reduce aquaculture production costs and prevent water quality deterioration resulting from excess feeding. In practice, sh are fed by most
farmers to satiety based on visual observation. However, it is very di cult to identify the point at which sh are satiated and sh are overfed frequently. The
stomach and intestine may exceed their capacity as a result of overfeeding, which leads to decreases in digestive e ciency and feed utilization [11]. On the
other hand, an insu cient 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 sh farming practices often result in sh stress and poor health. In contrast, improved feeding conditions not only bene t the welfare of sh but also
aquaculture pro ts [9]. In most sh species, high stocking densities adversely affect sh survival, weight gain, and speci c 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 sh 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 sh farming
practices affect sh physiology synergistically or individually.

2 Methods
2.1 Experimental diets
Diet formulation and proximate analysis [Association of O cial Analytical Chemists (AOAC), 1984][24] details are provided in Table 1. The diet ingredients
including shmeal, soy protein concentrate, casein, shrimp meal, wheat our, binding agents, soybean oil, sh 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 rst 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 sh and feeding trial
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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
sh farm (Hainan Chenhai Products Co., Ltd.). The sh 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 in ow 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 sh 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 sh per tanks, SD1.1), 2.2 kg m− 3 (1.1 kg sh per tank, SD2.2), 3.3 kg m− 3 (1.65 kg sh per tank, SD3.3), 4.4 kg m− 3 (2.2 kg sh per tank, SD4.4), and
5.5 kg m− 3(2.75 kg sh 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 sh 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 e ciency ratio (FER), speci c growth rate (SGR), survival, and
protein e ciency ratio (PER). Eight sh were randomly selected from each tank for proximate analysis, three for whole-body composition tests and ve for
white muscle components.

The viscera, liver, and intraperitoneal fat of sh 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 sh 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 × ( nal mean weight - initial mean weight)/initial mean weight (g)

Survival (%) = 100 ×  nal sh number/initial sh number

Average body weight gain (BWG, g) = (Wf− Wi)/number of shes;

Speci c growth ratio (SGR, % d−1) = 100 × (In Wf − Wi)/t;

Weight gain ratio (WGR) = (Wf− Wi)/Wi;

Daily feed intake (DFI, % d− 1) = 100 × dry feed intake/[(initial feed weight +  nal weight)/2 × t];

Condition factor (CF, g cm− 3) = 100 × (Wf/L3);

Hepatosomatic index (HIS, %) = 100 × (wet weight of the hepatopancreas/Wf);

Intraperitoneal ratio (IPR, %) = 100 × (intraperitoneal fat weigh/Wf);

where Wi and Wf are the initial and nal weights (g) of the sh during the experiment. t is the duration of the experiment (d); F is the weight of the feed
provided to the sh; L is the average body length (cm) of the sh.

2.5 Biochemical analysis

Proximate composition analyses of diets, sh 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 sh 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 nal 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 de ned 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 de ned as the release of 1 µmol of
fatty acids per minute per gram of protein. A slightly modi ed version of the Anson method was used to determine pepsin activity using casein as the

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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 xed with 10% buffered formalin for at least 24 hours, then dehydrated, para n-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 signi cant. 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. Wf,
BWG, WGR, SGR, and DFI were signi cantly different among the treatment groups (P < 0.05). Speci cally, Wf, BWG, WGR, SGR, and DFI in treatments G1, G7,
and G8 were signi cantly higher than those in the other treatments (P < 0.05). G7 had the highest Wf, BWG, WGR, and SGR, followed by treatments G1 and G8.
Our results indicated that optimal Wf, BWG, WGR, and SGR values were obtained at a stocking density of 2.2 kg m− 3 when the sh were fed twice a day,
followed by a 3.3 kg m− 3 stocking density when the sh were fed 3 times a day, relatively higher Wf, 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 signi cantly different even at high stocking densities and a 3-times-per-day feeding
frequency (Table 2). The DFI in the G3 group was signi cantly 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 sh 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 signi cant differences in CF were observed among groups except for G8, which exhibited a
signi cantly higher value, and G6 had the lowest value (P < 0.05). The HSI values of the sh in the G4 and G7 groups were signi cantly higher than those in
the G1, G2, G6, and G9 groups (P < 0.05) but not signi cantly different from those in other groups. Feeding frequency and stocking density had no signi cant
impact on sh, except in the G1 and G10 treatment groups.

According to the BWG regression model (y-axis) in response to the stocking density when the sh were fed twice a day (x-axis), the optimal stocking density
was 2.004 kg/m3. However, based on BWG (y-axis) in response to the stocking density when the sh were fed three times a day (x-axis), the optimal stocking
density was 1.343 kg/m3.

3.2 Biochemical analysis

3.2.1 Nutrients analyses in whole sh and muscle
As the stocking density increased in sh 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 signi cantly different from the G5 group (P > 0.05). In contrast, increasing the stocking density in sh fed
twice a day did not signi cantly affect moisture contents (P > 0.05). On the other hand, protein, fat, and ash contents were signi cantly different between
treatments, particularly in the G7 and G8 groups, which were signi cantly higher than the other three groups (P < 0.05).

When the stocking density was increased in sh fed three times a day, the moisture content of the whole sh was no signi cantly different (P > 0.05).
Moreover, the protein contents of G2-G5 and G9-10 were not signi cantly different (P > 0.05) but were signi cantly lower than those of the G1 and G6-7 groups
(P < 0.05). There was no signi cant difference in fat content (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 signi cantly 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 sh fed three times a day, AST (aspartate
aminotransferase) and CHO (cholesterol) levels increased gradually and were signi cantly different between the treatment groups, with the G1 group being
signi cantly lower than the other groups (P < 0.05). Moreover, when the sh were fed twice a day, the G7 and G8 groups were signi cantly 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 signi cantly different between treatment groups. When the sh 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
signi cantly higher GLU, LZM, SOD, and AKP values than the other groups. When the sh were fed twice a day, the G7 group exhibited signi cantly higher TP,

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GLB, and LZM values than the remaining groups (P < 0.05). The SOD and AKP values of groups G7-G9 were not signi cantly different, whereas G6 had the
highest values.

3.3 Tissues enzyme assays

As demonstrated in Table 5, there was a signi cant 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 signi cant 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 sh indicated the presence of in ammatory cells in these organisms.
Moreover, white vesicles were identi ed in the cells of sh 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 sh groups exhibited varying degrees of in ammation and fat granules
except for G7 and G8, which were normal. Figure 2 shows that increasing stocking densities in the sh fed three times a day resulted in lamina propria edemas
in the intestinal tissue sections of the G3, G4, and G5 sh, as well as thinner and shorter intestinal villi, some of which were starting to detach. In contrast, the
aforementioned symptoms were only identi ed in the G10 sh that were fed twice a day. Finally, as seen in Fig. 3, vacuolation occurred in the G2, G5, G6, G9,
and G10 sh, and nuclear deviations were observed in G3 and G4, whereas the hepatocytes in G1, G7, and G8 were normal.

4 Discussion
Many factors affect sh growth, including diet, sh size, feeding time, stocking density, and feeding frequency. In this experiment, when the sh were fed three
times a day, the BWG, WGR, and SGR of sh stocked at a density of 0.55 kg per tank gradually decreased with increasing stocking density and was
signi cantly higher than the other four groups. This suggested that the growth performance of the sh 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 in uenced 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 ndings 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 sh depending on the species. For instance,
Rowland et al. (2006) [30] reported that stocking density had no signi cant effects on silver perch. In contrast, high stocking density decreased the nal
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/m3,
whereas when the sh 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/m3.

Blood can objectively re ect 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. Speci cally, when liver cells are damaged, AST and ALT enzymes will in ltrate into the blood, causing a
signi cant 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 signi cantly with stocking density, except at a 2.2 kg/m3. Moreover, ALT decreased signi cantly with
increasing stocking density, particularly at 4.4 kg m− 3 and 5.5 kg m− 3 in both feeding frequencies. Our ndings 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 sh 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-speci c immune function. LZY and AKP are also important indicators of
non-speci c 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 sh immune function.

Previous studies have demonstrated that high stocking densities were associated with decreases in SOD activity in some sh [39, 40] and shrimps [41, 42].
LZY is known to disrupt the structure of the bacterial cell wall, and therefore LZY levels can directly re ect lytic capacity, as well as the impact of pathogens
and other environmental factors on sh 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
in ammation, 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 sh digestion and fat translation. Moreover, TP and ALB showed a gradual

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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 re ect the physiological status of sh 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 ngerlings were all depressed in high-density conditions (600 organisms m− 3) [44]. This indicated that the sh exhibited better digestive physiology
at an appropriate stocking density, which may also explain why the sh 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 signi cantly in uence gastric, intestinal, and hepatic tissue histology. When the sh 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 sh 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 sh 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/m3 (approximately 10 sh) when the sh were fed three times a day, and 2.004 kg/m3 (approximately 20–30 sh)
when the sh were fed twice a day.

Abbreviations
SD: stocking densities; FF: feeding frequencies; WGR: weight gain rate; SGR: speci c growth rate; FER: feed e ciency ratio; PER: protein e ciency 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 o cial
analytical chemists; MS222: tricaine methanesulfonate; SD: standard deviation; Wi : initial weights; Wf : nal 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 con icts 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 sh. All authors read and approved the nal manuscript.

-Acknowledgements

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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.

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Tables
Table.1 Formulation and proximate composition of the experimental diet.

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 Ingredients %

Fishmeal 50.00

Soy protein concentrate 12.00

Casein 4.00

Shrimp meal 3.50

Wheat our 20.5

Binding agents 2.00

Soybean oil 1.50

Fish oil 1.50

Squid visceral ointment 1.50

Vitamin premix2 1.00

Mineral premix3 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.

1. Obtained from Guangzhou Rifeng Science & Technology Co., LTD (Guangzhou, China).
2. Vitamin mixture (mg kg−1 diet): retinol acetate, 38.0; cholecalciferol, 13.2; a-tocopherol, 210.0; thiamin, 115.0; ribo avin, 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).
3. Mineral mixture (mg kg−1 diet): MgSO4·7H2O, 3568.0; NaH2PO4·2H2O, 25568.0; KCl, 3020.5; KAl (SO4)2, 8.3; CoCl2, 28.0; ZnSO4·7H2O, 353.0; Calactate,
15968.0; CuSO4·5H2O, 9.0; KI, 7.0; MnSO4·4H2O, 63.1; Na2SeO3, 1.5; C6H5O7Fe·5H2O, 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)

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 Group Growth performance Biometric indices

Wi (g) Wf (g) BWG(g) WGR (%) SGR(%/d) DFI (%/d) Survival(%) CF(g/cm3) HIS (%) IP

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

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

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

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

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

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

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

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

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

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

ANOVA (P value)

Breeding density < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 0.047 0.012 0

Feeding frequency < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 0.221 0.948 0

Interactions < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 0.032 0.003 0

                       

Values (mean ± S.D.) with different letters within the same row are signi cantly different (P<0.05). The absence of letters indicates no signi cant 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 (% WWa) 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 signi cantly different (P<0.05). The absence of letters indicates no signi cant difference
between treatments.

a WW: wet weight.

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Table.4 Serum biochemistry (mean ± S.D., n=3).

Group Items

AST ALT TP GLB ALB TG CHO GLU LZM

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

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

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

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

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

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

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

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

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

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

ANOVA(P value)

Breeding < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.00
density

Feeding < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.00
frequency

Interactions < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.00

                                   

Values (mean ± S.D.) with different letters within the same row are signi cantly different (P<0.05). The absence of letters indicates no signi cant 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).

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 Group Gastric Intestinal Hepatic

Pepsin Amylase Lipase (U/mg Pepsin Amylase Lipase Pepsin Amylase Lipase
(U/mg (U/mg prot) (U/mg prot) (U/mg (U/mg prot) (U/mg prot) (U/mg (U/mg prot)
prot) prot) prot) 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 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001
frequency

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 signi cantly different (P<0.05). The absence of letters indicates no signi cant difference
between treatments.

a WW: wet weight.

Figures

Figure 1

H&E stained gastric tissue sections of juvenile pearl gentian grouper. (A-J) Gastric tissue sections of sh in groups G1 to G10, respectively (H&E, 400)

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Figure 2

H&E stained intestinal tissue sections of juvenile pearl gentian grouper. (A-J) Intestinal tissue sections of sh in groups G1 to G10, respectively (H&E, 400)

Figure 3

H&E stained hepatic tissue sections of juvenile pearl gentian grouper. (A-J) Hepatic tissue sections of sh in groups G1 to G10, respectively (H&E, 400)

Figure 4
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Quadratic regression model based on body weight gain (y-axis) in response to sh stocking density when fed twice a day (x-axis).

Figure 5

Quadratic regression model based on body weight gain (y-axis) in response to sh stocking density when fed three times a day (x-axis).

Supplementary Files
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