Aquaculture Reports 26 (2022) 101303

Contents lists available at ScienceDirect

Aquaculture Reports
journal homepage: www.elsevier.com/locate/aqrep

Corn-starch based formulated diet has growth and feed utilization

efficiency advantages over trash fish diet for juvenile golden pompano
Trachinotus ovatus
Jun Wang a, b, c, Delbert M. Gatlin III d, Guangde Wu a, Yun Wang a, Chuanpeng Zhou a,
Heizhao Lin a, f, *, Zhenhua Ma a, c, e, *
a
Key Laboratory of Aquatic Product Processing, Ministry of Agriculture and Rural Affairs, South China Sea Fisheries Research Institute, CAFS, Guangzhou 510300, PR
China
b
Southern Marine Science and Engineering Guangdong Laboratory, Zhanjiang 524025, PR China
c
Sanya Tropical Fisheries Research Institute, Sanya 572018, PR China
d
Department of Ecology and Conservation Biology, Texas A&M University, College Station, TX 77843-2258, USA
e
Key Laboratory of Efficient Utilization and Processing of Marine Fishery Resources of Hainan Province, Sanya 572426, PR China
f
Shenzhen Base of South China Sea Fisheries Research Institute, CAFS, Shenzhen, PR China

A R T I C L E I N F O A B S T R A C T

Keywords: Until now, trash fish/low valued fish continues to be widely used in marine cage culture of pompano and many
Golden pompano other carnivorous species in China and other Asian countries. This study is the first attempt to compare the
Trachinotus ovatus performance of juvenile golden pompano Trachinotus ovatus fed trash fish or starch-based formulated diets. Three
Trash fish
isonitrogenous (41 %) and isolipidic (11 %) diets containing 16, 22 or 28 % corn starch were prepared. Juvenile
Formulated diet
Carbohydrate
pompano (mean initial weight: 28.0 ± 0.3 g) were fed either formulated diets or trash fish on a equivalent dry-
Corn starch matter basis for 8 weeks. The survival rates were significantly higher in fish fed starch diets than fish fed trash
Intestinal microbiota fish (P < 0.05). Specific growth rate (SGR) of fish fed the 22 % and 28 % starch diets was significantly higher
than fish fed 16 % starch diet and trash fish. Feed intake (FI) of fish fed trash fish was significantly higher than
those fed formulated diets; however, feed efficiency (FE) and protein efficiency ratio (PER) of fish fed the
formulated diets were higher than those fed trash fish, with the 22 % starch dietary group having a significantly
higher FE value than fish fed trash fish. The whole-body energy retention (ER) of fish fed the diet containing 22
% starch had the highest ER, significantly higher than those fed 16 % starch and trash fish. The moisture in
whole-body tissues of fish fed trash fish was significantly higher than the formulated dietary groups. On the other
hand, whole-body protein and lipid contents of fish fed the 22 % starch diet was significantly higher than the
other dietary treatments. The whole-body lipid level of fish fed trash fish was significantly lower than the other
dietary groups. The body condition indices were significantly higher for fish fed formulated diets compared to
those fed trash fish. The viscerosomatic index (VSI) was decreased with increasing dietary starch levels. Hexo­
kinase (HK) activity significantly increased with increasing dietary starch levels, whereas pyruvate kinase (PK)
activity had an opposite trend. The liver and muscle glycogen contents were generally increased with increasing
dietary starch levels. In addition, the intestinal microbial communities of fish fed the formulated diets and trash
fish was distinctly different. The results shown that formulated diet had advantages in performance and feed
utilization efficiency over trash fish. Corn starch can be used as a suitable dietary carbohydrate. Evaluated on
growth and FE, the optimal dietary corn starch level for T. ovatus is 22–24 % of diet.

1. Introduction 20 years with over 82 million tonnes of fish provided in 2020 (FAO,
2008). Providing cost-effective and sustainable feed input continues to
The production from aquaculture has grown dramatically in the last be the one of the most important factors in intensive aquaculture, as feed

* Corresponding authors at: Key Laboratory of Aquatic Product Processing, Ministry of Agriculture and Rural Affairs, South China Sea Fisheries Research Institute,
CAFS, Guangzhou 510300, PR China.
E-mail addresses: linheizhao@163.com (H. Lin), zhenhua.ma@hotmail.com (Z. Ma).

https://doi.org/10.1016/j.aqrep.2022.101303
Received 25 July 2022; Received in revised form 12 August 2022; Accepted 13 August 2022
Available online 17 August 2022
2352-5134/© 2022 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
J. Wang et al. Aquaculture Reports 26 (2022) 101303

represents approximately 60 % of total production costs. Table 1

Soluble carbohydrates are the least expensive forms of dietary energy Composition of experimental formulated diets (g/100 g dry weight) and trash
for fish and many other domestic animals. For fish, carbohydrate is fish.
generally not considered as an essential nutrient (Wilson, 1994), Formulated diets
whereas, various fish species can use soluble carbohydrates for meta­ corn starch level (%)
bolic energy (NRC, 2011). If soluble carbohydrates are not sufficient in Ingredients (% dry weight) 16 % 2% 28 % Trash fish
the diet, fish will catabolize protein and lipids for energy, which may Fishmeala 40.0 40.0 40.0 –
impair protein retention and increase the release of nitrogen into waters Soybean protein concentratea 19.3 19.3 19.3 –
Fish oil 6.0 6.0 6.0
(Cowey and Walton, 1989; Wilson, 1994; NRC, 2011). Therefore, under –
Lecithin 2.0 2.0 2.0 –
aquaculture conditions, provide appropriate levels of soluble carbohy­ Mineral premixb 1.8 1.8 1.8 –
drate in the diets can induce a protein-sparing effect (Wilson, 1994), and Vitamin premixc 2.0 2.0 2.0 –
can consequently lower the use of protein materials such as fishmeal and Choline chloride 0.4 0.4 0.4 –
soybean meal, which are more expensive than carbohydrate ingredients. Sodium alginate 0.5 0.5 0.5 –
Corn starch 16.0 22.0 28.0
Improved growth performance was found in many fish species including
–
Microcrystalline cellulose 12.0 6.0 0 –
tilapia Oreochromis niloticus (Anderson et al., 1984), Atlantic salmon Proximate composition
Salmo salar (Hemre et al., 1995), and cobia Rachycentron canadum (Ren Moisture 10.14 8.38 11.36 72.99
et al., 2001) when optimum levels of carbohydrates were included in Crude protein 40.6 41.1 40.6 68.4
Crude lipid 11.5 11.4 11.3 7.9
their diets. However, the abilities of fish to utilize dietary carbohydrate
Ash 13.9 13.4 13.2 14.7
varies among species and remains somewhat obscure. Optimal carbo­ Gross energy (kJ/g)d 16.9 18.0 18.8 19.3
hydrate level should be evaluated as too much carbohydrate in the diet a
Fishmeal: obtained from Russia. AKROS Fishing Co., Ltd. (Russia), Cod
can lead to negative effects on fish, including hyperglycemia, depressed
fishmeal, crude protein, 61.7 % dry matter, crude lipid, 6.6 % dry matter.;
growth, and lipid deposition in organs and fish carcass (Zhou et al.,
Soybean protein concentrate: 66.5 %, crude lipid, 0.52 %.
2015; Zhao et al., 2020; Cui et al., 2010). Thus, research on the nutri­ b
Mineral premix (mg or g/kg diet): NaF, 2 mg; KI, 0.8 mg; CoCl20.6 H2O (1
tional value of carbohydrate for various fish species is of great %), 50 mg; CuSO40.5 H2O, 10 mg; FeSO4⋅H2O, 80 mg; ZnSO4⋅H2O, 50 mg;
importance. MnSO4⋅H2O, 60 mg; MgSO40.7 H2O, 1200 mg; Ca (H2PO4)2⋅H2O, 8000 mg;
Golden pompano Trachinotus ovatus is a good candidate for intensive NaCl, 100 mg; Zoelite, 8485 mg; Ca (H2PO4)2⋅H2O, 1000 mg.
aquaculture, because of its good taste, fast growth, and hardiness in a c
Vitamin premix (mg or g/kg diet): thiamin, 25 mg; riboflavin, 45 mg; pyri­
crowded environment (Zhou et al., 2015). It has been widely cultured in doxine HCl, 20 mg; vitamin B12, 0.1 mg; vitamin K3, 10 mg; niacin acid, 200 mg;
China in recent years, with an annual production of ~110, 000 mt pantothenic acid, 60 mg; folic acid, 20 mg; biotin, 1.20 mg; retinal acetate, 32
(China Fishery Statistics Yearbook, 2021). However, trash fish (low mg; cholecalciferol, 5 mg; α–tocopherol, 120 mg; inositol, 800 mg; microcrys­
valued fish) is still widely used in culture of pompano and many other talline cellulose, 12,473 mg. Vitamin C, 200 mg.
d
Gross energy: calculated on the basis of protein, 23.6 kJ/g; lipid, 39.3 kJ/g;
carnivorous species in China and other Asian countries (Bunlipatanon
starch, 17.2 kJ/g (Shiau and Chen, 1993).
et al., 2014; Qi et al., 2019). This is controversial to the sustainability of
wild fisheries (Cao et al., 2015). Therefore, developing and using of
formulated feeds is necessary for sustainable aquaculture of these spe­ the diets were broken up and sieved into pellet with about 2-mm length
cies. However, till now, studies on the comparative efficiencies of trash and then stored at − 20 ◦ C until used.
fish and formulated feed are relatively scant (Tacon et al., 1991a; b; The species of trash fish used in the present study was Japanese scad
Usman et al., 2009; Shapawi et al., 2011; Bunlipatanon et al., 2014). (Decapterus maruadsi) which were chopped either manually or me­
The corn starch is one of the least expensive carbohydrates and chanically, into 2–3 mm cubes and stored at − 20 ◦ C until fed to the
commonly used in extruded feeds. In this study, three formulated diets experimental pompano.
with graded levels of corn starch and a trash fish feed were prepared and
fed to juvenile golden pompano Trachinotus ovatus. The performance, 2.2. Experimental procedure
feed utilization, metabolic enzyme activities, and intestinal microbiota
of juvenile pompano fed either formulated diets or trash fish were The feeding trial was conducted in a pond system of a seaside marine
compared to assess the feasibility and efficiency of corn starch as a laboratory, located at Shenzhen Basement of South China Sea Fisheries
carbohydrate source. Research Institute, Guangdong Province, southern China. Juvenile
T. ovatus were obtained from a local commercial hatchery. Upon arrival,
2. Materials and methods they were placed in a floating cage (3 (width, W) × 3 (length, L) × 3
(depth, D) m) and fed a commercial diet for marine fish (Guangdong
2.1. Diet preparation Haid Group, Guangzhou, China) for 2 weeks to acclimate them to
culturing conditions and feeding regime. At the beginning of the
Previous studies indicated the digestible carbohydrates in the diets of experiment, the fish were starved for 24 h, weighed, and then fish with
marine and coldwater fish species should not exceed 20 % (Kaushik and similar size (initial body wet weight 28.0 ± 0.3 g) were randomly and
Oliva-Teles, 1985; Beamish and Medland, 1986; Kaushik et al., 1989; evenly stocked in 12 outdoor cages (1.0 W × 1.0 L × 1.5 D m). Each cage
Hemre et al., 1995). Therefore, we designated three isonitrogenous and was stocked with 20 fish. Each experimental diet was randomly fed to
isolipidic diets (approximately 41 % crude protein and 11 % lipid, three separate groups of fish. Cages was provided with continuous
respectively) containing 16 %, 22 %, or 28 % corn starch in the diet aeration by two pond aerators.
(Table 1). Groups of fish were slowly hand-fed to satiation based on visual
Experimental diets were formulated from practical ingredients, using observation of their feeding behavior twice daily (06:30 and 16:30 h).
Russian steam-treated fishmeal and soy protein concentrate as the main The moisture content of the trash fish was measured regularly so that the
protein sources, menhaden oil as the lipid source. Ingredients were intake was computed on a dry-matter basis. Feed consumption was
mixed thoroughly with oil and distilled water to produce a stiff dough. recorded for each cage. The duration of the study was 8 weeks. Water
The dough was then cold-pelletized through a single-screw mincer (F-26 quality parameters were monitored daily. Water temperatures ranged
(II), South China University of Technology, Guangzhou, China) fitted from 27 to 31 ℃, salinity from 25 to 28 ppt, pH from 7.4 to 8.3,
with a 2 mm die, and the resulting noodle-like strings were dried at 25 ◦ C ammonia level was lower than 0.05 mg L− 1, and dissolved oxygen was
in an air-conditioned room with the aid of an electrical fan. After drying, >6.0 mg L− 1 throughout the experimental period.

2
J. Wang et al.
2.3. Sample collection and biochemical analysis
At the end of the feeding trial, fish were starved for 24 h and were
bulk-weighed. The total number of fish in each cage was recorded. Ten
fish were randomly sampled from each cage and ground into composite
samples to measure whole-body composition. Proximate composition
analysis of feed ingredients, experimental diets and fish were performed
according to AOAC methods (Association of Official Analytical Chem­
ists, 1995). Samples of the diets and fish were dried to a constant weight
at 105 ◦ C to determine moisture content. Protein was determined by
measuring nitrogen (N × 6.25) using the Kjeldahl method. Lipid levels
were determined by ether extraction using a Soxhlet apparatus. Ash
levels were measured by combustion at 550 ◦ C. Glycogen in the liver and
muscle samples was determined using a commercial kit (Nanjing Jian­
cheng Bioengineering Institute) following the manufacturer’s protocol.
Livers and whole intestine samples were removed and pooled from
three additional fish per cage for subsequent enzyme assay (n = 9 fish
per dietary treatment). Preparation of samples and the subsequent
enzymatic assays were followed the same procedure of a previous study
(Wang et al., 2019). All analyses were conducted in triplicate.
2.4. Intestinal bacteria incubation and sequencing
2.4.1. Characterization of the intestinal bacteria
At the end of the feeding trial, species composition of intestinal
microbiota communities of fish fed the diet with 22 % starch as well as
those fed trash fish, and the microbial composition of cultivation water
were examined. Three fish from each cage fed the 22 % starch and trash
fish diets were randomly sampled. The intestinal tract was aseptically
dissected and the intestinal contents were collected and inoculated
immediately onto TCBS (thiosulfate citrate bile saccharose) agar plates
at 28 ◦ C for 96. The dominant isolates were purified by streaking and re-
streaking onto fresh media. The following incubation and microbiolog­
ical examination including Gram staining were performed according to
Wang et al. (2013) and Dong and Cai (2001). Microbiota composition
analysis of cultivation water was performed with similar procedures.
2.4.2. PCR (polymerase chain reaction) and DGGE (denaturing gradient
gel electrophoresis)
The digestive tracts from three fish per cage were aseptically
dissected and the intestinal contents were collected, pooled, and flash-
frozen in liquid N2 and stored at − 20 ◦ C until analyzed. The pooled
intestinal samples were Genomic DNA was isolated from the initial in­
testinal content sample as described Rosales et al. (2017). PCR was
conducted using the method of Hume et al. (2003) with bacteria-specific
PCR primers 518R (5′ -ATTACC GCGGCTGCTGG-3′ ) and 338F (5′ -CCT
ACG GGA GGC AGC AG-3′ ) to conserved regions flanking the variable
V3 region of 16S rDNA. The PCR amplifications were performed on a
Biometra Tprofessional Basic 96 Gradient, Thermocycler (Analytik Jena
AG, Jena, Germany). The PCR product was sequenced by Shanghai
Sangon company (China). The sequences were aligned and compared
with available sequences in the NCBI GenBank using BLAST. The band
pattern relatedness was analyzed using Molecular Analysis Finger­
printing software (v1.6; Bio-Rad, Hercules, CA, USA) based on the Dice
similarity coefficient (SC) and the unweighted pair group method.
Comparisons between band patterns of samples were expressed as a
percentage SC (SC > 95 %, 80 % ≤ SC ≤ 95 %, and SC < 80 % indicate
two populations are identical, similar; and significantly different,
respectively).
2.5. Calculations and statistical analysis
Growth (measured as specific growth rate (SGR: %⋅day− 1)), feed
intake (FI, g 100 g− 1 BW (body wet weight) day− 1), feed efficiency (FE),
protein efficiency ratio (PER), condition factor (CF), hepatosomatic
index (HSI), viscerosomatic index (VSI), intraperitoneal fat (IPF) ratio,
3
Aquaculture Reports 26 (2022) 101303
and energy retention (ER) were calculated as described previously
(Wang et al., 2022).
Results were analyzed by one-way analysis of variance (ANOVA).
When the ANOVA identified differences among dietary groups, multiple
comparisons with Duncan’s multiple range test were performed among
means. Significance was set at P < 0.05. Normality and homoscedas­
ticity assumptions were confirmed prior to any statistical analysis.
Second-order polynomial regression (Zeitoun et al., 1976) was used to
estimate the relationship between SGR and FE and dietary corn starch
levels, and the optimal level of corn starch in diet for juvenile T. ovatus
was aslo estimated. All tests were performed in SPSS 18.0 for windows
(IBM Corp., Armonk, N.Y., USA).
3. Results
3.1. Survival and performances
Dietary starch levels had significant influences on the survival,
growth, and feed intake of golden pompano (Table 2). The survival rates
of fish fed starch diets were relatively high (91–95 %), and significantly
(P < 0.05) higher than that of fish fed trash fish. Specific growth rate
(SGR) of fish fed the 22 % and 28 % starch diets was significantly (P <
0.05) higher than fish fed the 16 % starch diet and trash fish. No sig­
nificant difference was found between 22 % and 28 % dietary treatments
(P > 0.05). The feed intake (FI) of fish fed trash fish was significantly (P
< 0.05) higher than that of fish fed formulated diets. No significant
difference exist among the three starch-based dietary treatments. The
feed efficiency (FE) and protein efficiency ratio (PER) of fish fed the
formulated diets were higher than that fed trash fish, with fish fed 28 %
starch diet having the highest FE value, significantly (P < 0.05) higher
than those fed trash fish. The energy retention (ER) showed a similar
trend of FE, with fish fed the 22 % starch diet having the highest ER,
significantly (P < 0.05) higher than the 16 % starch diet and trash fish
groups.
When second-order polynomial regression analysis was employed,
based on SGR and FE to estimate the optimal dietary starch level for
T. ovatus, the regression equations were: Y = − 0.0068 X2 + 0.32 X −
1.15 (R2 = 0.60) and Y = − 0.01 X2 + 0.46 X − 3.73 (R2 = 0.44),
respectively (Fig. 1). The estimated optimum dietary corn starch level
for juvenile T. ovatus was 23.9 % and 22.2 %, respectively.
3.2. Whole-body composition and body condition indices
The moisture in whole-body tissues of fish was highest in fish fed
trash fish, significantly (P < 0.05) higher than those fed the formulated
diets. Fish fed the diet with 22 % starch had the lowest moisture,
significantly lower than the other dietary treatments (Table 3). On the
other hand, body protein and lipid contents was highest in fish fed 22 %
starch diet, significantly higher than the other dietary treatments. The
whole-body lipid content of fish fed trash fish was significantly lower
than the other dietary groups. Whereas, the ash content of pompano fed
trash fish was significantly higher than fish fed the diets containing 16 %
and 28 % starch (Table 3).
The body condition indices were significantly affected by the dietary
treatments, they were all significantly (P < 0.05) higher for pompano
fed formulated diets than those fed trash fish (Table 3). In regard to fish
fed the formulated diets, no significant differences in body condition
indices existed among the groups. Fish fed the diet with 22 % starch
group had the highest condition factor (CF); whereas, The viscer­
osomatic index (VSI) decreased with increasing dietary starch levels.
Hepatosomatic index (HSI) and intraperitoneal fat (IPF) ratio were
similar among fish fed the three formulated diets, with that of the 22 %
starch group a bit lower (Table 3).
J. Wang et al. Aquaculture Reports 26 (2022) 101303

Table 2
Performance and feed utilization of juvenile golden pompano Trachinotus ovatus fed with corn-starch-based formulated diets or trash fish for 8 weeks.
Diet (starch Final body Survival Specific growth rate Feed intake (g⋅100 g Feed Protein efficiency Energy
content, %) weight (g) (%⋅d− 1) BW⋅d− 1), efficiency ratio retention

16 % 101.5 ± 1.3b 91.1 ± 2.32 ± 0.04b 2.28 ± 0.18b 0.99 ± 2.43 ± 0.32a 45.0 ± 7.5a
2.2a 0.13ab
22 % 130.6 ± 8.8a 95.6 ± 2.73 ± 0.11a 1.85 ± 0.16b 1.38 ± 0.30b 3.35 ± 0.71a 73.8 ± 14.2b
2.1a
28 % 124.7 ± 10.4a 91.1 ± 2.66 ± 0.14a 2.04 ± 0.28b 1.01 ± 2.49 ± 0.68a 54.5 ± 4.1ab
1.2a 0.28ab
Trash fish 82.5 ± 0.8c 82.2 ± 1.95 ± 0.01c 3.33 ± 0.21a 0.59 ± 0.13a 0.86 ± 0.20b 19.4 ± 1.9c
2.0b

Values are means of three replicate groups ± SEM. Values in the same column with same letters are not significantly different (one-way ANOVA and Duncan’s multiple-
range tests, P > 0.05).

(P < 0.05) decreased with increasing dietary starch levels.

The liver glycogen increased with increasing dietary starch levels,
but the differences among dietary treatments did not reach a significant
level (P > 0.05). The muscle glycogen was higher in golden pompano
fed the diets with 22 % and 28 % starch than those fed 16 % starch
(Table 4).

3.4. Intestinal microbiota

The number and dominant species of intestinal microbiota of juve­

Fig. 1. The effects of dietary corn starch levels on (A) specific growth rate
(SGR) and (B) feed efficiency (FE) of juvenile golden pompano Trachinotus
ovatus. Each point represents the mean (±SEM) of three replicate groups of fish
for each dietary treatment.
3.3. Carbohydrate digestive and metabolic enzyme activities, and
glycogen contents
No significant difference existed in amylase, GK, and PFK activities
among fish fed all the starch dietary treatments (P > 0.05) (Table 4).
However, HK activity significantly (P < 0.05) increased with increasing
dietary starch levels. On the other hand, the PK activities significantly
nile pompano were markedly influenced by dietary treatments. The
number of intestinal bacterial colonies of fish fed with trash fish was
7.8 × 105 cfu/g, higher than that of fish fed the diet with 22 % starch
(3.0 × 105 cfu/g). Both were higher than the number of bacterial col­
onies in culture water (2.5 ×104/g mL).
The intestinal bacteria of fish fed the diet with 22 % starch, and the
bacteria of culture water were primarily Gram negative bacteria. The
dominant species were Pasteurella spp and Flavimonas oryzihabitans,
respectively. Both Gram negative and positive bacteria (Bacillus) were in
the gastrointestinal tract (GIT) of fish fed trash fish, with Flavimonas
oryzihabitans and Bacillus sphaericus as the dominant species (Table 5).
Dietary treatments resulted to distinct differences in intestinal mi­
crobial communities in juvenile golden pompano. Nineteen species were
identified, including fifteen species belong to Proteobacteria, two spe­
cies of Firmicutes, one species of Actinobacteria, and one species of
fusobacterium. There were distinct differences in 16S rDNA polymerase
chain reaction (PCR) products among fish fed the different diets (Fig. 2).
Fish fed the diets containing 22 % and 28 % starch had the most similar
microbial profiles which were distinctly different from that of fish fed
the trash fish and the diet containing 16 % starch diet (Fig. 2).
4. Discussion
In the present study, survival of experimental golden pompano fed
either trash fish or formulated diets were relatively high. The growth
rates of juvenile golden pompano fed formulated diets were comparable
to previous studies with fish of a similar initial body size (Zhou et, 2015;
Wang et al., 2019; Zhao et al., 2020), indicating the nutritive values of
the experimental diets were able to meet the requirements of the
experimental fish. The growth rates (SGR), feed efficiencies (FE), protein

Table 3
Whole-body composition and body condition indices including condition factor (CF), hepatosomatic index (HSI), viscerosomatic index (VSI), intraperitoneal fat (IPF)
ratio, of golden pompano Trachinotus ovatus fed the experimental diets.
Diet (starch content, %) Moisture Crude protein Crude lipid Ash CF VSI HSI IPF ratio

16 % 68.9 ± 0.80b 18.0 ± 0.23c 10.3 ± 0.08b 3.56 ± 0.11b 3.57 ± 0.05b 6.52 ± 0.19a 1.00 ± 0.05a 0.82 ± 0.11a
22 % 63.2 ± 0.19c 21.1 ± 0.09a 13.8 ± 0.71a 4.14 ± 0.14a 3.76 ± 0.05a 6.43 ± 0.22a 0.95 ± 0.03a 0.81 ± 0.06a
28 % 67.7 ± 0.06b 18.8 ± 0.31b 11.3 ± 0.41b 3.59 ± 0.07b 3.73 ± 0.09ab 6.21 ± 0.13a 1.00 ± 0.07a 0.82 ± 0.08a
Trash fish 71.3 ± 0.09a 18.3 ± 0.16bc 6.8 ± 0.10c 4.06 ± 0.03a 3.37 ± 0.05c 5.16 ± 0.23b 0.77 ± 0.06b 0.48 ± 0.05b

Values are means of three replicate groups + SEM. Values in the same column with same letters are not significantly different (one-way ANOVA and Duncan’s multiple-
range tests, P > 0.05).

4
J. Wang et al. Aquaculture Reports 26 (2022) 101303

Table 4
Carbohydrate digestive and metabolic enzyme activities and glycogen contents of juvenile golden pompano Trachinotus ovatus fed experimental diets containing
graded levels of corn starch for 8 weeks.
Diet (starch content, %) Amylase Glucokinase Phosphofructokinase-1 Hexokinase Pyruvate kinase Liver glycogen (mg/g) Muscle glycogen (mg/g)
(U/mg prot) (mU/L) (U/mL) (U/g prot) (U/g prot)

16 % 3.87 ± 0.76 14.79 ± 0.31 6.46 ± 0.35 200.8 ± 8.96a 92.4 ± 5.90a 4.37 ± 0.77 1.36 ± 0.12b
22 % 3.66 ± 0.84 14.49 ± 0.06 6.82 ± 0.34 276.1 ± 14.94b 88.8 ± 3.75ab 4.91 ± 0.26 1.86 ± 0.13a
28 % 3.72 ± 0.08 14.32 ± 0.45 6.63 ± 0.25 482.5 ± 10.45c 71.8 ± 3.46bc 4.94 ± 0.19 1.66 ± 0.16ab

Values are means of three replicate groups ± SEM. Means in each column with different superscripts have significant differences (one-way ANOVA and Duncan’s
multiple-range tests, P > 0.05).

Table 5
The dominant species of culturable microbiota in the intestinal tract of fish fed
22 % starch based formulated diet and trash fish, and in culture water.
Sample Gram stain microscopy Dominant species
Culture Gram negative bacteria Pasteurella spp
water
22 % starch Gram negative and positive Flavimonas oryzihabitans/
bacteria /Bacillus Bacillus sphaericus
Trash fish Gram negative bacteria Flavimonas oryzihabitans
efficiency ratio (PER), and energy retention (RE) of pompano fed
formulated diets were significantly higher than that of fish fed trash fish.
These results clearly demonstrated that juvenile pompano can effec­
tively utilize formulated dry pelleted feeds.
Until now, very limited studies compared the performance of fish fed
trash fish and formulated diets. Bunlipatanon et al. (2014) compared
performances of Asian seabass Lates calcarifer and tiger grouper Epi­
nephelus fuscoguttatus fed with either trash fish or compounded feeds in
cage aquaculture system. They concluded that for both species, the
overall survival and growth rates for the two feeds were similar. How­
ever, in our study, the growth rate of golden pompano fed formulated
diets were higher than that of fish fed trash fish. The different obser­
vations could be due to several reasons, e.g., differences in species
composition of trash fish, nutritive values of formulated diets, culture
operations and feeding procedures, and inter-specific differences in feed
acceptance, etc. The better performance of formulated diets could be
due to that they can provide a more balanced profile of nutrients than
trash fish. Furthermore, compared to the dry formulated diets, trash fish
are more susceptible to pathological bacteria during storage, and
correspondingly, more likely to transmit disease during feeding. These
could be also possible reasons for the lower survival of golden pompano
fed trash fish.
In regard to feed efficiency, calculated on the data from Bunlipata­
non et al. (2014), the FE were ~0.47 and ~0.86 for commercial pellet
feed (CPF) and trash fish fed Asian seabass, respectively, and ~0.37 and
~0.30 for CPF and trash fish fed tiger grouper, respectively. In the
present study, the FE of fish fed corn-starch based formulated diets
ranged from 0.99 to 1.38, and was approximately 0.59 for trash fish. The
FE from the present study was comparable with those from Bunlipata­
non et al. (2014), and is in agreement with Qi et al. (2019), who reported
that for fish fed with trash fish in floating cage culture in Daya Bay (the
same area where the feeding trial of the present study was carried out)
was approximately 0.57. Therefore, the better performance and higher
feed utilization efficiency of formulated dry feed are evident. Formu­
lated diet has advantages over trash fish as feed types for pompano. In
addition, the use of dry formulated pellet feed is much more easier to
store, particularly, it does not need daily preparatory procedures prior to
feeding as trash fish does. This is another potential advantage of
formulated feed over trash fish (Tacon et al., 1991a,b; Bombeo-Tuburan
et al., 2001).
Source of carbohydrates is a key factor influencing their utilization
efficiencies (Stone, 2003). In the present study, performance and FE
suggested that golden pompano utilized corn starch well. Moreover,
higher PER and body protein deposition were induced in fish fed the
starch diets compared to those fed trash fish. This could be due to uti­
lization of carbohydrate inhibiting amino acid catabolism, thereby
having a protein-sparing effect. Similarly, Zhou et al. (2015) fed juvenile
golden pompano diets with graded levels of carbohydrate (0–28 %), and
found that the PER increased as dietary carbohydrate increased from
0 % to 16.8 %. These results have important implications, because sol­
uble carbohydrates are the least expensive source of dietary energy for
fish, and among them corn and wheat starch are good binders and the
most widely used in practical aquatic feeds (Hertrampf and
Piedad-Pascal, 2000). However, it is noteworthy that body lipid content,
CF, VSI, HSI, and IPF of fish fed formulated diets were higher than fish
fed trash fish, indicating a higher deposition of lipid in fish body.
Similarly, Zhou et al. (2015) found that the lipid contents in whole-body
and dressed carcass of golden pompano increased as the dietary carbo­
hydrate level increased from 0 % to 11.2 %. Zhao et al. (2020) also found
that the HSI of juvenile golden pompano linearly increased with
increasing dietary carbohydrate levels. Rawles and Gatlin, 1998 found
that increase the levels of soluble carbohydrate resulted to a higher HSI
and IPF ratios as well as liver glycogen in sunshine bass M. chrysops ×
M. saxatilis and striped bass Morone saxatilis. When salmonids were fed
diets with high levels of digestible carbohydrates, their liver size and
glycogen content were proportionally increased to the dietary carbo­
hydrate levels (Bergot, 1979; Hilton and Atkinson, 1982; Enes et al.,

Fig. 2. Dendrogram of microbial samples from juvenile golden pompano fed trash fish or formulated diets containing different percentages of corn starch.

5
J. Wang et al.
2008; Moreira et al., 2008). Zhou et al. (2015) also found that the muscle
glycogen contents of T. ovatus were increased significantly with
increasing dietary carbohydrate levels. In the present study, we found
that the liver and muscle glycogen contents were higher in fish fed the
diets with 22 % and 28 % starch compared to those fed the 16 % starch
diet. Therefore, in the present study the liver enlargement may result
from either increased lipid or glycogen deposition, or both.
Previous studies indicated that the digestible carbohydrates in the
diets of marine or coldwater fish species should not higher than 20 %
(Kaushik and Oliva-Teles, 1985; Beamish and Medland, 1986; Kaushik
et al., 1989; Hemre et al., 1995). In a previous study by Zhou et al.
(2015) the optimal dietary carbohydrate of golden pompano was esti­
mated to be 12.1 % based on maximum SGR. However, in the present
study, based on SGR and FE, we estimated that the optimal dietary corn
starch level for juvenile pompano was 22–24 % of diet. The lower value
in Zhou et al. (2015) could be due to their diets not being isolipidic, the
dietary lipid varied from 4.6 % to 16.8 %. It has been demonstrated that
other nutrients, such as lipid and protein, fiber, and dietary energy, and
their ratios to carbohydrate all can influence the utilization of dietary
carbohydrates (Shiau, 1997). Also, experimental fish with different body
size were used. In our study, the experimental fish had a larger initial
body size (averaged 28.0 g) than that in the previous study (initial body
weight 9.24 g). In terms of the influence of body size on carbohydrate
utilization, Tung and Shiau (1993) conducted a study on tilapia with two
body sizes (average weight: 4.55 g, 0.46 g), they found that larger fish
had significantly higher growth rate, FCR, and protein and energy
deposition than the smaller ones when fed a glucose diet. These results
suggested that fish with larger body size has a higher carbohydrate
utilization ability, and could tolerate a higher dietary carbohydrate level
compared to fish with smaller body size. This could be another possible
reason for the higher estimated value in the present study.
The abilities to utilize carbohydrate varied among species and
depend on their carbohydrate digestive enzyme activities (Stone et al.,
2003). The major enzymes for carbohydrate digestion are present in fish
(Cowey and Walton, 1989). In the present experiment, the activities of
hepatic amylase, GK, and PFK-1 did not vary markedly with the gradient
levels of dietary carbohydrate, suggesting that they might not be critical
for regulating carbohydrate utilization in golden pompano. In mammals,
two hepatic enzymes, hexokinase (HK) and glucokinase (GK), serve as
key enzymes in the regulation of blood glucose. In the present study, the
hexokinase (HK) activity markedly increased as dietary starch levels
increased; whereas, pyruvate kinase (PK) activity were markedly
decreased as dietary carbohydrate levels increased. Similarly, Zhou et al.
(2015) fed juvenile golden pompano with diets containing graded levels
of carbohydrate, and found that the HK activity significantly increased
as dietary starch level increased from 0 % to 16.8 % and then nearly
plateaued. HK plays a major role in the nutritional regulation of the
glycolytic pathways (Walton and Cowey, 1982; Panserat et al., 2001).
Results from the present study suggested that endogenous glycolysis in
juvenile T. ovatus was enhanced when starch was present in the diet.
Theoretically, the intestinal microbiota in the gastrointestinal tract
of fish merely come from cultivation water before the first feeding. After
feeding, the bacteria come from both cultivation water and feed. Until
now, very few studies reported the influences of diet on the intestinal
microbiota of golden pompano (Zhao et al., 2020). The present study for
the first time compared the intestinal microbiota species composition of
juvenile T. ovatus fed trash fish and starch-based formulate diets. We
observed that the intestinal microbiota communities altered in many
respects. The obvious differences in bacterial numbers, species’
composition, and dominant species of culturable microbiota in the
gastrointestinal tract among fish fed starch-based formulated diet and
trash fish, and culture water clearly demonstrated that the feed types are
important factors shaping the intestinal microbial communities. None­
theless, the intestinal microbiota of fish is still fairly poorly known.
Further specific studies are needed to elucidate the regulation mecha­
nism of feed on intestinal microbiota communities of T. ovatus.
6
Aquaculture Reports 26 (2022) 101303
In conclusion, our results indicated that formulated diet had some
advantages in performance and feed utilization efficiency over trash
fish. Corn starch can be used as a suitable dietary carbohydrate, and its
optimal level for juvenile pompano is 22–24 % of diet.
CRediT authorship contribution statement
Jun Wang: Conceptualization, Methodology, Supervision, Writing –
review & editing, Funding acquisition. Delbert M. Gatlin III: Manu­
script editing and revision, Guangde Wu: Feeding experiment, sam­
pling, Yun Wang: Data analysis, Chuanpeng Zhou: Chemical analysis.
Heizhao Lin: Feed preparation, Zhenhua Ma: Feeding experiment.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
No data was used for the research described in the article.
Acknowledgements
The work was supported by grants from the National Natural Science
Foundation of China (32172984), the earmarked fund for CARS-47,
Central Public-interest Scientific Institution Basal Research Fund,
CAFS (2020TD55, 2020TD29, 2022XT0404), Central Public-interest
Scientific Institution Basal Research Fund, South China Sea Fisheries
Research Institute, CAFS (2021SD09, 2021XK02), the National Key
Research and Development Program of China (2018YFD0900703), the
Fund of Southern Marine Science and Engineering Guangdong Labora­
tory (Zhanjiang) (ZJW-2019-06), and Key Laboratory of Efficient Utili­
zation and Processing of Marine Fishery Resources of Hainan Province.
References
Anderson, J., Jackson, A.J., Matty, A.J., Capper, B.S., 1984. Effects of dietary
carbohydrate and fiber on the tilapia Oreochromis niloticus (Linn.). Aquaculture 37,
303–314.
AOAC (Association of Official Analytical Chemists), 1995. Official Methods of Analysis,
16th ed. AOAC International, Washington, DC.
Beamish, F.W.H., Medland, T.E., 1986. Protein sparing effects in large rainbow trout,
Salmo gairdneri. Aquaculture 55, 35–42.
Bergot, F., 1979. Carbohydrate in rainbow trout diets: effects of the level and source of
carbohydrate and the number of meals on growth and body composition.
Aquaculture 18, 157–167.
Bombeo-Tuburan, I., Coniza, E.,B., Rodriguez, E.M., Agbayani, R.F., 2001. Culture and
economics of wild grouper (Epinephalus coioides) using three feed types in ponds.
Aquaculture 201, 229–240.
Bunlipatanon, P., Songseechan, N., Kongkeo, H., Abery, N.W., De Silva, S.S., 2014.
Comparative efficacy of trash fish versus compounded commercial feeds in cage
aquaculture of Asian seabass (Lates calcarifer) (Bloch) and tiger grouper (Epinephelus
fuscoguttatus) (Forsskål). Aquacult. Res. 45, 373–388.
Cao, L., Naylor, R., Henriksson, P., Leadbitter, D., Metian, M., Troell, M., Zhang, W.,
2015. China’s aquaculture and the world’s wild fisheries. Science 347, 133–135.
Cowey, C.B., Walton, M.J., 1989. Intermediary metabolism. In: Halver, J.E. (Ed.), Fish
Nutrition, 2nd edn..,. Academic Press, New York, pp. 259–329.
Cui, X.J., Zhou, Q.C., Liang, H.O., Yang, J., Zhao, L.M., 2010. Effects of dietary
carbohydrate sources on the growth performance and hepatic carbohydrate
metabolic enzyme activities of juvenile cobia (Rachycentron canadum Linnaeus.).
Aquacult. Res. 42 (1), 99–107.
Dong, X.Z., Cai, M.Y., 2001. Manual for Identification of Common Bacterial. Science
Press, Beijing.
Enes, P., Panserat, S., Kaushik, S., Oliva-Teles, A., 2008. Growth performance and
metabolicutilization of diets with native and waxy maize starch by gilthead sea
bream (Sparus aurata) juveniles. Aquaculture 274, 101–108.
FAO (Food and Agriculture Organization), 2008. FAO yearbook: fishery and aquaculture
statistics. Rome.
Hemre, G.I., Sandnes, K., Lie, Ø., Torrissen, O., WaagbØ, R., 1995. Carbohydrate
nutrition in Atlantic salmon, Salmo salar L.: growth and feed utilization. Aquacult.
Res. 26, 149–154.
J. Wang et al.
Hertrampf, J.W., Piedad-Pascal, F., 2000. Handbook on Ingredients For Aquaculture
Feeds (Marine oils), vol. 281. Kluwer Academic Publishers, Dordrecht, the
Netherlands, pp. 338–349.
Hilton, J.W., Atkinson, J.L., 1982. Response of rainbow trout (Sulmo gairdneri) to
increased levels of available carbohydrate in practical trout diets. Brit. J. Nutr. 47,
597–607.
Kaushik, S.J., Oliva-Teles, A., 1985. Effect of digestible energy on nitrogen and energy
balance in rainbow trout. Aquaculture 50, 89–101.
Kaushik, S.J., Médale, F., Fauconneau, B., Blanc, D., 1989. Effect of digestible
carbohydrates on protein/energy utilization and on glucose metabolism in rainbow
trout (Salmo gairdneri R.). Aquaculture 79, 63–74.
Moreira, I.S., Peres, H., Couto, A., Enes, P., Oliva-Teles, A., 2008. Temperature and
dietary carbohydrate level effects on performance and metabolic utilization of diets
in European sea bass (Dicentrarchus labrax) juveniles. Aquaculture 274, 153–160.
NRC (National Research Council), 2011. Nutrient requirements of fish and shrimp.
National Academy Press, Washington, DC.
Panserat, S., Capilla, E., Gutierrez, J., Frappart, P.O., Vachot, C., Plagnes-Juan, E.,
Aguirre, P., Brèque, J., Kaushik, S., 2001. Glucokinase is highly induced and glucose-
6-phosphatase poorly repressed in liver of rainbow trout (Oncorhynchus mykiss) by a
single meal with glucose. Comp. Biochem. Physiol. B: Biochem. Mol. Biol. 128,
275–283.
Qi, Z.H., Shi, R.J., Yu, Z.H., Han, T.T., Li, C.H., Xu, S.M., Xu, S.N., Liang, Q.Y., Yu, W.,
Lin, H.Z., Huang, H.H., 2019. Nutrient release from fish cage aquaculture and
mitigation strategies in Daya Bay, southern China. Mar. Pollut. Bull. 146, 399–407.
Rawles, S.D., Gatlin III, D.M., 1998. Carbohydrate utilization in striped bass Morone
saxatilis and sunshine bass M. chrysops × M. saxatilis. Aquaculture 161, 201–212.
Ren, M.C., Ai, Q.H., Mai, K.S., Ma, H.M., Wang, X.J., 2001. Effect of dietary carbohydrate
level on growth performance, body composition, apparent digestibility coefficient
and digestive enzyme activities of juvenile cobia, Rachycentron canadum L. Aquacult.
Res. 42 (10), 1467–1475.
Shapawi, R., Mustafa, S., Ng, W.K., 2011. A comparison of the growth performance and
body composition of humpback grouper (Cromileptes altivelis) fed on farm made
feeds, commercial feeds or trash fish. J. Fish. Aquat. Sci. 6, 523–534.
Shiau, S.Y., 1997. Utilization of carbohydrates in warmwater fish-with particular
reference to tilapia, Oreochromis niloticus × O. aureus. Aquaculture 151, 79–96.
Shiau, S.Y., Chen, M.J., 1993. Carbohydrate utilization by Tilapia (Oreochromis niloticus
× O. aureus) as influenced by different chromium sources. J. Nutr. 123, 1747–1753.
Stone, D.A.J., 2003. Dietary carbohydrate utilization by fish. Rev. Fish. Sci. 11 (4),
337–369.
Aquaculture Reports 26 (2022) 101303
Stone, D.A.J., Allan, G.L., Anderson, A.J., 2003. Carbohydrate utilization by juvenile
silver perch, Bidyanus bidyanus (Mitchell). III. The protein-sparing effect of wheat
starch-based carbohydrates. Aquacult. Res. 34, 123–134.
Tacon, A.G.J., Rausin, N., Kadari, M., Cornelis, P., 1991a. The food and feeding of marine
in floating cages at the National Seafarming Development Centre, Lampung,
Indonesia: rabbit fish, Siganus canaliculatus (Park). Aquacult. Fish. Manag. 21,
375–390.
Tacon, A.G.J., Rausin, N., Kadari, M., Cornelis, P., 1991b. The food and feeding of
tropical marine fishes in floating net cages: Asian seabass, Lates calcarifes (Bloch),
and brown spotted grouper. Epinephelus tauvina (Forskal). Aquacult. Fish. Manag. 22,
165–182.
Tung, P.H., Shiau, S.Y., 1993. Carbohydrate utilization versus body size in tilapia
Oreochromis niloticus × O. aureus. Biochem. Physiol. 104A (3), 585–588.
Usman, R., Palinggi, N.N., Williams, K., 2009. Formulated feed for tiger grouper grow-
out. Aquac. Asia Mag. 30–35.
Walton, M.J., Cowey, C.B., 1982. Aspects of intermediary metabolism in salmonid fish.
Comp. Biochem. Physiol. B: Comp. Biochem. 73, 59–79.
Wang, J., Xu, H.G., Zuo, R.T., Mai, K.S., Ai, Q.H., 2022. Chromium polynicotinate
inclusion in the diet affects growth, feed utilization, and chromium retention in
Japanese seabass Lateolabrax japonicas fed a low protein starch-based diet.
Aquaculture 560, 738569.
Wang, J., Gatlin III, D.M., Li, L.H., Wang, Y., Niu, J., Lin, H.Z., Zhou, C.P., Huang, Z.,
Yu, W., Guo, Y.J., 2019. Dietary chromium polynicotinate improves growth
performance and feed utilization of juvenile golden pompano (Trachinotus ovatus)
with starch as the carbohydrate. Aquaculture 505, 405–411.
Wang, R.X., Feng, J., Su, Y.L., Ye, L.T., Wang, J.Y., 2013. Studies on the isolation of
Photobacterium damselae subsp. piscicida from diseased golden pompano (Trachinotus
ovatus Linnaeus) and antibacterial agents sensitivity. Vet. Microbiol. 162, 957–963.
Wilson, R.P., 1994. Utilization of dietary carbohydrate by fish. Aquaculture 124, 67–80.
Zeitoun, I.H., Ullrey, D.E., Magee, W.T., 1976. Quantifying nutrient requirement of fish.
J. Fish. Res. Board Can. 33, 167–172.
Zhao, W., Xie, J.J., Fang, H.H., Liu, Y.J., Tian, L.X., Niu, J., 2020. Effects of corn starch
level on growth performance, antioxidant capacity, gut morphology and intestinal
microflora of juvenile golden pompano, Trachinotus ovatus. Aquaculture 524,
735197.
Zhou, C.P., Ge, X.P., Niu, J., Lin, H.Z., Huang, Z., Tan, X.H., 2015. Effect of dietary
carbohydrate levels on growth performance, body composition, intestinal and
hepatic enzyme activities, and growth hormone gene expression of juvenile golden
pompano, Trachinotus ovatus. Aquaculture 437, 390–397.