Aquaculture 548 (2022) 737718

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Aquaculture
journal homepage: www.elsevier.com/locate/aquaculture

Effects of different feeding frequencies on the growth, plasma biochemical

parameters, stress status, and gastric evacuation of juvenile tiger puffer fish
(Takifugu rubripes)
Xiao Qiang Gao a, Xi Wang a, b, Xin Yi Wang a, b, Hong Xu Li a, Liang Xu a, Bin Huang a,
Xue-Song Meng c, Tao Zhang c, Hai-Bin Chen d, Rui Xing d, Bao-Liang Liu a, *
a
Key Laboratory for Sustainable Development of Marine Fisheries, Ministry of Agriculture, Qingdao Key Laboratory for Marine Fish Breeding and Biotechnology, Yellow
Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, 266071, People’s Republic of China
b
College of Fisheries and Life Science, Ocean University, Shanghai, 201306, People’s Republic of China
c
Dalian Tianzheng Industrial Co. Ltd., Dalian, 116000, People’s Republic of China
d
Yuhai Hongqi Ocean Engineering Co. LTD, Rizhao, 276800, People’s Republic of China

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

Keywords: Tiger puffer fish (Takifugu rubripes)is one of the most economically valuable aquaculture species in Asia. How­
Takifugu rubripes ever, farming tiger puffer fish at the juvenile stage has been greatly restrained by cannibalization caused by
Feeding frequency inappropriate feeding regimes, resulting in very high mortality and great economic loss, particularly in intensive
Growth performance
recirculating aquaculture systems. Therefore, the feeding strategy should adapt to fish appetite, nutrition re­
Oxidative stress
Gastric evacuation
quirements and welfare in intensive aquaculture. A 60-day feeding experiment was conducted to evaluate the
effects of feeding frequency on the overall health of juvenile tiger puffer fish. Fish were randomly assigned to
four groups with different feeding frequencies using the same ration size per feeding. P1: two daily meals; P2:
four daily meals; P3: six daily meals; P4: continuous diurnal feeding using a belt feeder. Fish in the P2 group
exhibited optimal values for growth, including final weight, weight gain rate, specific growth rate, feed effi­
ciency, and survival (P < 0.05), followed by those in the P3 group. The crude lipid content increased significantly
with increasing feeding frequency (P < 0.05), indicating that high frequency feeding could accumulate higher
body fat content. The apparent digestibility coefficients of dry matter, protein, and lipid were significantly lower
for fish in the P4 group than for those in the other groups (P < 0.05). The total cholesterol, triglyceride, and
glucose levels increased significantly with increasing feeding frequency (P < 0.05), indicating that high fre­
quency feeding leads to excessive accumulation of dietary carbohydrates and lipids in juvenile tiger puffer fish.
Fish in the P1 and P4 groups exhibited relatively higher antioxidant enzyme activities (P < 0.05), which sug­
gested that low and high feeding frequencies cause oxidative stress in juvenile tiger puffer fish. A comparison of
non-linear model parameters revealed that an exponential model best described the gastric evacuation rate of
juvenile tiger puffer fish, with approximately half the meal passed within 3 h, which further confirmed that
feeding intervals in four times/day may more closely align with the digestive physiological characteristics of
juvenile tiger puffer fish. Overall, these results indicate that the best growth performance and physiological
indicators can be achieved when fed at 4 times/day.

1. Introduction
Feeds and feeding constitute the main costs in intensified aquacul­
ture. Rational feed management can significantly decrease these costs
and improve the profitability of fish farming. Feed management includes
variables such as food size and type, feeding rate, feeding frequency, and
delivery techniques (Kousoulaki et al., 2015; Xie et al., 2011). Among
them, feeding frequency is one of the most important factors and has
attracted much attention from aquaculture researchers (Salgado-Ism­
odes et al., 2020). Optimizing feeding frequency may not only promote
better growth, lower feed conversion, and increased survival, but may
also minimize feed wastage, contribute to improved water quality, and/

* Corresponding author.
E-mail address: liubl@ysfri.ac.cn (B.-L. Liu).

https://doi.org/10.1016/j.aquaculture.2021.737718
Received 26 July 2021; Received in revised form 11 November 2021; Accepted 12 November 2021
Available online 19 November 2021
0044-8486/© 2021 Elsevier B.V. All rights reserved.
X.Q. Gao et al.
or reduce size coefficients of variation (Cho et al., 2003; Biswas et al.,
2006; Villarroel et al., 2011; Cunha et al., 2013). Moreover, inadequate
feeding frequencies reportedly reduce fish growth, increase cannibalism
rates, and oxidative damage, and even depress immunity, resulting in
significant economic losses (Li et al., 2014; Tian et al., 2015; Guo et al.,
2018; Sousa et al., 2019). Therefore, optimum feeding frequencies have
been investigated in a wide range of commercially important fish species
(Lee et al., 2000a; Wu et al., 2018; Gilannejad et al., 2019; Hu et al.,
2020; Gilannejad et al., 2020; Mabroke et al., 2021).
The analysis of hematological parameters is an important practice in
evaluating fish welfare, for they reflect physiological conditions such as
stress and health (Barton, 2000; Tavares-Dias and Moraes, 2007; Kita­
gawa et al., 2015). It has been previously reported that feeding fre­
quency affected the changes of blood parameters to varying degrees. For
example, blood parameters, e.g. plasma glucose and cholesterol, can
change due to feeding frequency (Rosenlund et al., 2004). Oh et al.
(2018) found that as feeding frequency increased, the plasma total
cholesterol and high-density lipoprotein cholesterol contents also
significantly increased. According to Busti et al. (2020), most of the
plasma parameters related to nutritional and physiological conditions
were not affected by feeding frequency, however an effect on electro­
lytes (Na+, Cl), cortisol and creatinine was observed. Al-Khafaji et al.
(2017) also reported that plasma glucose and cholesterol were signifi­
cantly higher for fish fed three times a day compared with once a day.
Thus, different feeding frequency may influence a number of physio­
logical variables, and it would seem useful to understand the conse­
quences of feeding practices.
Gastric evacuation models based on stomach content data are useful
for quantifying food consumption rates (Khan et al., 2016). The term
gastric evacuation denotes the rate at which food passes from the
stomach into the intestine, reflecting stomach fullness at postprandial
time, especially at return of appetite, which helps researchers and
farmers estimate daily rations and energy budgets of fish (Jia et al.,
2020). Booth et al. (2008) reported that feeding fish at peak appetite
may improve growth and feed digestion efficiency. Thus, gastric evac­
uation rate (GER) experiments have been used to optimize feeding re­
gimes for farmed fish. Previous studies have reported that GER depends
on numerous variables, including size of the feed grain, dietary energy
density, fish size, temperature, and prey resistance to the digestive
process in the stomach (Andersen, 2001; John et al., 2004; Justin et al.,
2013; Khan et al., 2016; Kim et al., 2017). Studying GER under different
feeding frequencies is also crucial for optimizing the growth and
development of fish.
Tiger puffer fish (T. rubripes), known as the ‘king of fish’, is the most
commercially valuable and highly traded fish species in Asia, especially
in Japan, Korea, and China, due to its white, tender, and tasty meat
(Zhou et al., 2019). Unfortunately, the natural stocks of tiger puffer fish
have greatly declined in the last few decades because of environmental
pollution and habitat destruction. To protect wild resources and meet
market demand, guidelines for mass culture of this species have been
established in China, including conditions for egg quality, nutritional
requirements, nitrite toxicity, growth, and light (Wei et al., 2020; Gao
et al., 2019; Wei et al., 2019; Liu et al., 2019; Zhao et al., 2018). Pro­
duction has increased rapidly with the gradual expansion of tiger puffer
aquaculture on the northern coast of China in recent years, reaching
approximately 25,000 tons in 2018. However, at present, farming tiger
puffer fish at the juvenile stage has been greatly restrained by canni­
balization, particularly in intensive recirculating aquaculture systems. It
has been reported that the phenomenon of cannibalism is closely related
to feeding frequency. Muntaziana et al. (2017) found that no mortalities
occurred over 42 days when Channa striatus fry were fed twice a day,
compared to a 80% survival rate in the eight times a day feeding
treatment, which clearly indicates that an increase in feeding frequency
can lead to the cannibalism to some extent. Kaiser et al. (1995) reported
that African catfish, Clarias gariepinus larvae were more aggressive in
between feedings when higher feeding frequencies were adopted.
2
Aquaculture 548 (2022) 737718
However, one study found that lower feeding frequencies increased the
aggressive behavior of Limanada ferruginea juveniles (Dwyer et al.,
2002). Therefore, investigating correlations between feeding regimes
and healthy farming of this species is urgently needed. Here, we aimed
to analyze the effects of different feeding frequencies on the growth,
plasma biochemical parameters, stress status, and gastric evacuation of
juvenile tiger puffer fish. The study findings provide new insights to help
decrease cannibalism rates of juvenile tiger puffer fish, thus improving
aquaculture outcomes of this species.
2. Materials and methods
2.1. Fish and experimental design
Juvenile T. rubripes were obtained from Tangshan MuHai Aquacul­
ture Co. Ltd. (Hebei, China).
The fish were acclimatized in the indoor cement pools (6.0 m × 6.0
m × 1.2 m) for two weeks before starting the experiment. The stocking
density was set as 1.68 kg/m3. During the acclimation period, the fish
were fed a commercial diet (HaiTong Group Foods, Fujian, China) twice
a day at 2.0% their body weight (size 3.0–4.0 mm). Proximate compo­
sition of the experimental diets is presented in Table 1. Half of the water
was renewed every 24 h. The water conditions were maintained as fol­
lows, temperature: 23.3 ◦ C–25.7 ◦ C, salinity: 29–31 ppt, dissolved ox­
ygen: 7.23–8.57 mg/L, pH: 7.26–7.84. Afterwards, 1200 healthy fish of
similar size (average initial weight 15.58 ± 0.19 g) were randomly
distributed in a closed recirculating aquaculture system equipped with
twelve circular tanks (diameter 1.0 m, volume 1000 L). Each tank
contained 100 fish with three replicate tanks per experimental group.
Fish were fed the mentioned above commercial diet using the following
feeding regimes: Group P1 received two daily meals at 06:30 and 18:30
h; Group P2 received four daily meals at 06:30, 10:30, 14:30 and 18:30
h; Group P3 received six daily meals at 06:30, 08:54, 11:18, 13:42, 16:06
and 18:30 h; and Group P4 received continuous diurnal feeding using a
belt feeder (Fig. 1). Fish were fed to apparent satiation, which was
assessed prior to the experimental period, as described by Gilannejad
et al. (2019). The daily ration (4.00% body weight of fresh tiger puffer
fish) was equally distributed to all meals during the multiple feeding
process. The food consumption was recorded daily. Uneaten pellets were
collected using automatic filter collection equipment, immediately
placed in a drying chamber for 12 h at 80 ◦ C, and weighed. The fish were
observed daily and mortalities were recorded. During the feeding
experiment, the sand-filtered seawater within each tank was set at a
flowrate of 10 L/min to ensure suitable water conditions. The average
water temperature was 24.0 ± 1.1 ◦ C, the pH was 7.56 ± 0.21, the
dissolved oxygen content was maintained at 7.84 ± 0.52 mg/L, the
salinity was 29–31 ppt, the total ammonia level was 0.28 ± 0.03 mg/L,
and the nitrite level was 0.36 ± 0.03 mg/L.
2.2. Sample collection
The experimental period was 60 days from July 15 to September 15,
2020. The study consisted of three trials that were performed consecu­
tively on the same fish stock: trial 1 – apparent digestibility; trial 2-
Table 1
Proximate composition of the experimental diets (% on a wet weight basis).
Treatments P1 P2 P3 P4
Moisture 4.81 5.13 4.85 4.93
Crude protein 53.21 53.15 53.04 53.25
Crude lipid 12.11 12.14 12.16 12.13
Ash 8.94 9.03 9.11 8.97
Note: P1, P2, P3 and P4 refer to groups fed for two daily meals, four daily meals,
six daily meals and continuous diurnal feeding, respectively.
X.Q. Gao et al. Aquaculture 548 (2022) 737718

06:30

18:30
Feeding time
P1
Sampling time

03:00

07:00
17:00

23:00
13:00

19:00
10:00

15:00
12:00
09:00
08:00

11:00
07:00

18:30
06:30

10:30

14:30
Feeding time
P2
Sampling time

23:00
15:00

07:00
12:00

13:00

17:00

03:00
10:00

19:00
11:00
07:00

08:00

09:00

16:06

18:30
08:54

11:18
06:30

13:42

Feeding time
P3
Sampling time
15:00

19:00

23:00
13:00

17:00

07:00
10:00
08:00

12:00
11:00
09:00

03:00
07:00
06:30

18:30

Feeding time
P4
Sampling time
19:00

23:00
17:00
13:00

15:00
12:00
10:00

03:00
11:00
07:00

09:00
08:00

07:00
Fig. 1. Detailed schematic of the daily feeding protocols and sampling schedule for juvenile tiger puffer, Takifugu rubripes. P1, P2, P3 and P4 refer to groups fed for
two daily meals, four daily meals, six daily meals and continuous diurnal feeding, respectively. White and black bars indicate light and dark periods.

growth and related physiological indicators; and trial 3- gastrointestinal replicate tank, a total of 15 fish from each treatment group, were
evacuation. The experiments were carried out in strict accordance with randomly selected. Blood samples were quickly drawn from the caudal
the Guide for the Care and Use of Laboratory Animals of Chinese vessel using a 2 mL syringe. The serum was obtained by centrifugation at
Academy of Fishery Sciences. The procedures were approved by the 12,000g for 5 min at 4 ◦ C, and then stored at − 80 ◦ C for further analysis.
Committee on the Ethics of Animal Experiments of Chinese Academy of Additionally, the livers were individually removed and immediately
Fishery Sciences. weighed to determine the hepatosomatic index (HSI). Subsequently,
three fish from each replicate tank were randomly collected and stored
2.2.1. Trial 1 – Apparent digestibility at − 80 ◦ C for whole-body proximate composition analysis.
On the last 15 trial days, the apparent digestibility coefficients
(ADCs) were determined to evaluate the influence of feeding frequency 2.2.3. Trial 3 – Gastrointestinal filling and evacuation
on the digestive abilities of juvenile tiger puffer fish. Yttrium oxide At the end of trials 1 and 2, the remaining fish were used to deter­
(Y2O3), used as an inert marker, was evenly added to the above com­ mine the gastrointestinal evacuation model. Juveniles were fed the
mercial diet at a concentration of 2%. On day 46 of the 60-day experi­ commercial diet for three days under the same feeding protocol (Fig. 1).
mental period, the marked diet was provided until the end of the On day 4, fish began to consume a single meal at 06:30 h with contin­
experiment. The feeding schedules and experimental conditions uous feeding for 30 min until apparent satiation, and the uneaten pellets
remained the same. Feces were collected at 1 h intervals by syphoning were retrieved. Sampling was performed at thirteen sequential time
the bottom of the tanks after each meal. The wet samples were centri­ points (0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, 16, 20 and 24 h) after the feed
fuged at 3000g for 20 min, the supernatant was removed, and the change in each protocol (Fig. 1). At each sampling time point, three fish
sediment was dried in a DHG electrothermal thermostatic blast oven from each circular tank were sacrificed. The abdominal cavity was
(Shanghai Longyue Instrument Equipment Co., Ltd., Shanghai, China) at dissected and the digestive tract was ligated at the esophagus, pylorus,
60 ◦ C for 24 h. The dried samples were stored at − 20 ◦ C until analysis. and anus using surgical clamps to prevent the contents of the abdominal
cavity from flowing out. The stomach contents included all material
2.2.2. Trial 2 – Growth and related physiological indicators contained between the posterior end of the esophagus and the anterior
At the end of the experiment (60 days), all fish were starved for 24 h, end of the small intestine. According to the method described by
and then were anesthetized with 50 mg/L tricaine methane sulfonate Nikolopoulou et al. (2011), the stomach contents were carefully
(MS-222, Sigma-Aldrich, St. Louis, MO, USA). Five fish from each collected in pre-weighed glass bottles (5 mL), weighed, freeze-dried,

3
X.Q. Gao et al. Aquaculture 548 (2022) 737718

reweighed, and the measured weights were used for further calculation. HSI = 100 × (liver weight/body weight)

2.3. Analytical methods 2.3.5. Gastric evacuation

2.3.1. Biochemical indicators
The concentrations of total protein (TP), albumin (Alb), globulin
(Glo), glucose (Glu), triglycerides (TG), and total cholesterol (TC) in
serum were measured using a PUZS-300 automatic biochemical
analyzer (Nanjing Perlong Medical Equipment Co. Ltd., Shanghai,
China) with commercial kits (Nanjing Jiancheng Bioengineering Insti­
tute, Nanjing, China), according to the method described by Jun et al.
For gastric evacuation, the geometric mean of the stomach digesta
was regressed against time in order to examine the possible fit of a model
for calculating gastric evacuation rate (GER). For each feeding protocol,
the feed residence time was estimated as the time during which the
content in the intestine was equal to or higher than 50% of the maximum
amount. In addition, the relative stomach or intestinal content (g dry
matter per 100 g BW) was determined by the following calculation:

Relative content = (dry matter removed from either the stomach or the intestine (g)/wet body weight of fish (g) ) × 100

(2015). The enzyme activities of superoxide dismutase (SOD), catalase

(CAT), and glutathione peroxidase (GPx) in serum were determined
using commercial kits (Mlbio, Shanghai, China) in accordance with the 2.4. Statistical analyses
manufacturer’s instructions. One unit of SOD activity was defined as the
amount of enzyme required to inhibit the oxidation reaction by 50%, All statistical analysis was performed using IBM SPSS v24.0 software
expressed as units per mg protein. One unit of CAT or GPx activity was (IBM Corp., Armonk, NY, USA). Experimental data were expressed as the
defined as the amount of enzyme required to consume 1 μmol of sub­ mean ± standard deviation (SD). The normality and homogeneity of
strate or generate1μmol of product per minute, respectively, expressed variance assumptions were verified using Kolmogorov-Smirnov and
as units per mg of soluble protein. The glutathione (GSH) content was Levene’s tests, respectively. The data were analyzed via one-way anal­
determined following the method described by Lin et al. (2018). ysis of variance (ANOVA, time). If significant differences were found, a
Tukey’s post-hoc test was used to identify the differences between
2.3.2. Apparent digestibility of dry matter, protein, and lipid means. The significance level was set to 0.05; differences were consid­
The apparent digestibility coefficients (ADCs) of dry matter, protein, ered significant if P < 0.05.
and lipid for diet and feces were calculated using the formula described
by Gilannejad et al. (2019): 3. Results
( )
dietary Y2 O3 level fecal nutrient level
ADC (%) = 100 × 1 − × The growth performance characteristics and survival rates of juve­
fecal Y2 O3 level dietary nutrient level
nile tiger puffer fish subjected to different feeding frequencies are shown
in Table 2. Fish in the P2 group exhibited the highest final weight, WGR,
2.3.3. Proximate composition of diet, feces, and fish
SGR, PER, LER, survival rate, and lowest FCR, followed by those in the
Diets, feces, and fish were analyzed in triplicate for proximate
P3 group. Among the abovementioned parameters, fish in the P4 group
composition following the AOAC (2000) procedure. Dry matter was
exhibited the worst values for growth performance and feed conversion
determined gravimetrically, weighing the sample after it achieved
ratio, followed by those in the P1 group. DFI was similar in all groups (P
constant weight after drying in an oven at 105 ◦ C. Ash content was
> 0.05). HSI was significantly higher in the P4 group than in the P1
determined by incinerating the sample in a muffle furnace at 550 ◦ C for
5 h. Crude protein content was measured using the Kjeldahl method (N
× 6.25). Crude fat content was determined by ether extraction. Table 2
The effect of feeding frequency on the growth performance of juvenile tiger
puffer, Takifugu rubripes reared under different feeding regimes for 60 days.
2.3.4. Growth parameters
Growth performance was assessed as relative growth rate (RGR, %), Treatments P1 P2 P3 P4

specific growth rate (SGR, %⋅d− 1), feed conversion ratio (FCR), daily IBW (g) 15.62 ± 0.10 15.33 ± 0.08 15.58 ± 0.12 15.78 ± 0.09
feed intake (DFI), protein efficiency ratio (PER, g/g), lipid efficiency FBW (g) 72.80 ± 1.54a 80.91 ± 1.54b 78.92 ± 1.95b
67.74 ±
1.43c
ratio (LER, g/g), survival rate (SR, %) and hepatosomatic index (HSI, %)
366.00 ± 427.90 ± 406.70 ± 329.18 ±
using the following equations (Kesbiç et al., 2020; Busti et al., 2020; Eya RGR (%)
12.49a 11.07b 15.52b 6.99c
et al., 2013; Yiğit et al., 2012). SGR (%/d) 2.56 ± 0.04a 2.77 ± 0.04b 2.71 ± 0.04b 2.43 ± 0.03c
DFI (g/fish) 0.82 ± 0.07 0.70 ± 0.05 0.69 ± 0.06 0.80 ± 0.07
RGR = 100 × [(final wet weight − initial wet weight)/initial wet weight ] FCR 1.14 ± 0.03a 0.93 ± 0.06b 0.95 ± 0.08b 1.26 ± 0.07c
PER (g/g) 1.65 ± 0.02a 2.02 ± 0.04b 1.98 ± 0.05b 1.49 ± 0.02c
SGR = 100 × [(ln final wet weight − ln initial wet weight)/days ] LER (g/g) 7.24 ± 0.06a 8.96 ± 0.21b 8.77 ± 0.16b 6.61 ± 0.12c
14.89 ± 15.38 ± 16.91 ±
HSI (%) 13.19 ± 1.10a
FCR = feed intake/weight gain 1.47ab 0.55ab 0.45b
Survival 82.00 ±
77.00 ± 3.51a 89.00 ± 2.67b 90.00 ± 1.90b
(%) 1.38a
DFI = (feed intake/number of fish)/day
Note: IBW, initial body weight (g); FBW, final body weight (g); RGR, relative
PER = (final wet weight − initial wet weight)/protein intake growth rate; SGR, specific growth rate; DFI, daily feed intake (g/fish); FCR, feed
conversion ratio; PER, protein efficiency ratio (g/g); LER, lipid efficiency ratio
LER = (final wet weight − initial wet weight)/lipid intake (g/g); FCR, feed conversion ratio; HSI, hepatosomatic index. P1, P2, P3 and P4
refer to groups fed for two daily meals, four daily meals, six daily meals and
SR = 100 × (final numbers of fish/initial numbers of fish) continuous diurnal feeding, respectively. Different superscript letters in each
row indicate significant differences between values (P < 0.05).

4
X.Q. Gao et al. Aquaculture 548 (2022) 737718

Table 3 feeding. The relative contents of the stomach were basically digested
The effect of feeding frequency on the proximate composition of juvenile tiger after 8 h, and digestion was completed in 10–12 h.
puffer, Takifugu rubripes reared under different feeding regimes for 60 days (%, GER was estimated by fitting the data recorded between 0.5 and 24 h
wet weight). (Fig. 5), as described by Andersen (2001), Nikolopoulou et al. (2011)
Treatments P1 P2 P3 P4 and Gilannejad et al. (2019). The collected data best fit an exponential
Moisture 75.73 ± 2.12 75.47 ± 2.54 76.81 ± 2.46 74.27 ± 1.67 model, expressed by the following formula (Elliott, 1972):
Crude protein 14.75 ± 0.58 15.73 ± 0.95 15.35 ± 0.47 14.43 ± 0.95 rt
Crude lipid 4.43 ± 0.08a 4.72 ± 0.09b 4.91 ± 0.07c 5.27 ± 0.05d
Wt = Ae−
Ash 2.62 ± 0.15 2.56 ± 0.29 2.50 ± 0.16 2.42 ± 0.18
Where Wt is the geometric mean weight of stomach digesta at time t,
Note: Different superscript letters within a row indicate significant differences A is the constant calculated from the regression model, and r is the rate
between values (P < 0.05). P1, P2, P3 and P4 refer to groups fed for two daily of gastric evacuation.
meals, four daily meals, six daily meals and continuous diurnal feeding, Feeding frequency did not affect GER of juvenile tiger puffer fish, as
respectively. shown by the similar fitted equations and models for each feeding group
displayed in Fig. 5. The gastric evacuation time (GET) was calculated as
described by Nikolopoulou et al. (2011), estimated from the semi-
Table 4
logarithmic model lnWt = lnA–rt. GET was computed as the time (t)
The effect of feeding frequency on the apparent digestibility of dry matter,
protein, and lipid in juvenile tiger puffer, Takifugu rubripes reared under different
required to empty half of the stomach contents. Feeding frequency did
feeding regimes for 60 days (%). not affect GET of juvenile tiger puffer fish, as shown in Table 5. GER,
predicted by the rate constant (r) in the global model, was approxi­
Treatments P1 P2 P3 P4
mately 5.10 ± 0.10 g per 100 g BW per min. Juvenile tiger puffer fish
Dry matter 76.14 ± 72.12 ± 75.77 ± 52.83 ± required approximately 180.62 ± 9.78 min (almost 3 h) to pass 50% of
digestibility 1.36a 4.41a 3.06a 2.55b
their ingested meal through the gastric chamber.
92.55 ± 93.97 ± 91.53 ± 78.88 ±
Protein digestibility
2.54a 3.27a 3.64a 3.01b
95.31 ± 94.94 ± 95.11 ± 84.03 ± 4. Discussion
Lipid digestibility
3.29a 1.26a 3.84a 2.35b

Note: Different superscript letters within a row indicate significant differences
between values (P < 0.05). P1, P2, P3 and P4 refer to groups fed for two daily
meals, four daily meals, six daily meals and continuous diurnal feeding,
respectively.
group (P < 0.05), but no significant differences were observed between
the P2 and P3 groups (P > 0.05).
The effects of different feeding frequencies on the proximate
composition of juvenile tiger puffer fish are reported in Table 3. The
moisture, crude protein, and ash contents of fish were not affected by
feeding frequency (P > 0.05). However, the crude lipid content of fish
increased significantly as feeding frequency increased (P < 0.05).
The effects of different feeding frequencies on the apparent di­
gestibility of dry matter, protein, and lipids in juvenile tiger puffer fish,
represented as ADC values, are listed in the Table 4. The ADC values of
dry matter, protein, and lipid for fish in the P4 group were significantly
lower than those for fish in the other groups (P < 0.05). However, no
differences were found among the other feeding groups (P > 0.05).
Changes in the serum constituents of juvenile tiger puffer fish sub­
jected to different feeding frequencies are presented in Fig. 2. Feeding
frequency did not affect the serum TP, Alb, and Glo contents (P>0.05).
Serum TC, TG, and Glu levels increased significantly with increasing
feeding frequency (P < 0.05), but no significant differences were found
between the P2 and P3 groups (P > 0.05).
The significant effects of feeding frequency on the activities of
antioxidant enzymes are shown in Fig. 3. SOD and CAT activities in the
P1 and P4 groups were significantly higher than those in the P2 and P3
groups (P < 0.05), but no significant difference was found between the
P1 and P4 groups, or between the P2 and P3 groups (P > 0.05). The
highest activities of GSH and GPx were found in the P4 group, followed
by the P1 group (P < 0.05), but the P2 and P3 groups did not differ
significantly (P > 0.05).
The passage of feed (dry matter) through the stomach organ of ju­
venile tiger puffer fish fed a single meal at 06:30 h is illustrated in Fig. 4.
Feeding frequency did not affect the dynamics of gastrointestinal
emptying for juvenile tiger puffer fish. Indeed, the overall stomach
content pattern was similar for the four different feeding regimes. The
relative stomach contents of initial satiation in feeding groups P1-P4
were 4.88 ± 0.18, 4.64 ± 0.18, 4.92 ± 0.19 and 4.94 ± 0.25 g per
100 g BW, respectively, which was drastically reduced within 2–4 h of
In the present study, fish in the P2 group (four times/day) displayed
the highest final weight, WGR, SGR, PER, LER, survival rate, and lowest
FCR, followed by those in the P3 group (six times/day). The results
indicated that a feeding frequency of 4–6 times daily could induce
optimal growth of juvenile tiger puffer fish (14.67 ± 1.16 g). The effect
of optimal feeding frequency on fish growth performance has been re­
ported in several species. Guo et al. (2018) reported that the optimal
feeding frequency to enhance growth and boost immunity of juvenile
Dolly Varden char (Salvelinus malma) (average weight 9.40 ± 0.30 g)
was four times/day. Oh et al. (2018) suggested that juvenile dark-
banded rockfish (Sebastes inermis) weighing 10–40 g can achieve opti­
mum growth performance when they are fed twice daily. Further, Silva
et al. (2007) reported that the best feeding strategy for tambaqui
(Colossoma macropomum) (average weight 2.67 ± 1.13 g) during the first
growth phase in cages was 10% BW/day divided between three meals/
day. Similarly, Al-Khafaji et al. (2017) also found that Jade Perch
Scortum barcoo can get the best growth and feeding efficiencies as well as
minimizing size variability in a recirculating system when they are fed
three times a day. These results likely indicate that the optimum feeding
frequency varies depending on fish species, size, and rearing condition.
In addition, much lower and higher feeding frequencies have adverse
effects on the growth and weight gain of fish. In our study, we found that
various growth parameters of fish in the P1 group (two times/day) were
significantly lower than those of fish in the P2 and P3 groups. Ewerton
et al. (2020) reported that low feeding frequency could not meet the
nutritional requirements for growth of juvenile liza (Lebranche mullet),
which concurred with the results of our study, which indicated that low
frequency feeding does not supply enough food to maintain healthy fish
growth, thus leading to malnutrition. Likewise, we found that excessive
feeding (P4 group, continuous diurnal feeding) exhibited the worst
values for growth performance and feed conversion ratio. These results
concurred with the study by Guo et al. (2018), in which higher feeding
frequency (six times/day) did not lead to an improvement in fish growth
performance. Higher feeding frequency not only caused food to move
through the digestive tract more quickly, resulting in reduced nutrient
absorption and digestive efficiency, but fish fed continuously displayed
greater swimming activity, which may have led to higher energy
expenditure (Johansen and Jobling, 1998; Liu and Liao, 1999; Dwyer
et al., 2002; Tian et al., 2015).
In the current study, the moisture, crude protein, and ash contents of
juvenile tiger puffer fish were not significantly affected by the different

5
X.Q. Gao et al. Aquaculture 548 (2022) 737718

A B
60 30

50 25

40 20

Alb (g/L)
TP (g/L)

30 15

20 10

10 5

0 0
P1 P2 P3 P4 P1 P2 P3 P4

C D
35 8

30 7
6 c
25 b

TC (mmol/L)
b
Glo (g/L)

5
20 a
4
15
3
10
2
5 1
0 0
P1 P2 P3 P4 P1 P2 P3 P4

E F
14 3

12
TG (mmol/L)

10 c
Glu (mg/dL)

2
c b
8 b b b
6 a a

1
4

0 0
P1 P2 P3 P4 P1 P2 P3 P4

Fig. 2. Biochemical blood parameters of juvenile tiger puffer, Takifugu rubripes reared under different daily feeding protocols for 60 days. These parameters include
total protein (TP, A), albumin (Alb, B), globulin (Glo, C), total cholesterol (TC, D), triglycerides (TG, E) and glucose (Glu, F). P1, P2, P3 and P4 refer to groups fed for
two daily meals, four daily meals, six daily meals and continuous diurnal feeding, respectively. Values are expressed as the mean ± SD. Different letters indicate
significant differences between groups at P < 0.05.

feeding regimes. However, the body lipid content was significantly
increased with increasing feeding frequency. Similar results were
observed with large yellow croaker (Larimichthys crocea) (Xie et al.,
2011), juvenile Korean rockfish (Sebastes schlegelii) (Lee et al., 2000a and
b), post-larval ayu (Plecoglossus altivelis) (Cho et al., 2003), and Brazilian
sardine (Sardinella brasiliensis) (Baloi et al., 2016). Studies found that
excess energy could be a reflection of increased whole-body protein or
lipid (Lee and Pham, 2010; Wu et al., 2015). A previous study reported
that excessive diet intake can result in a large amount of lipids being
stored in adipose tissues and the liver (McCue, 2010). In addition, we
found that HSI increased significantly with increasing feeding fre­
quency, which may indicate that the increased lipid intake was even­
tually converted into hepatic fat. Therefore, increased lipid levels in fish
may contribute to accumulation of fat in the liver. However, Al-Khafaji
et al. (2017) reported that increasing feeding frequencies beyond once a
day tended to increase the whole-body crude protein, while the lipid
content tended to be lower for Jade Perch fed three times a day
compared with the other treatments. These findings indicated that there
may be some differences in metabolic patterns under the different
feeding frequencies.
Lower digestibility levels were found for juvenile tiger puffer fish in
the P4 group; however, no differences were found among fish in the
other groups. This indicated that a fast gut-filling rate and shorter feed
residence time could result in significantly reduced digestion and ab­
sorption efficiency, with evacuated feed retaining relatively higher
levels of unabsorbed nutrients. This was further verified by the poor
growth performance and nutrient utilization efficiency of fish in the P4
group. Low digestibility levels could also be associated with low enzyme
to substrate ratios in the gut. The importance of the enzyme to substrate
ratio in final hydrolysis yield has been previously reported in several

6
X.Q. Gao et al. Aquaculture 548 (2022) 737718

A B
100 40
90 35
80
a 30 a

CAT (U/mgprot)
SOD (U/mgprot)

70 a
25 a
60 b b
50 20 b b
40 15
30
10
20
10 5
0 0
P1 P2 P3 P4 P1 P2 P3 P4

C D
40 40
35 35
30 30
GSH (U/mgprot)

GPx (U/mgprot)
25 25
a c
20 b b 20 a
b b
15 15
10 10
5 5
0 0
P1 P2 P3 P4 P1 P2 P3 P4

Fig. 3. Antioxidant enzymes activities of juvenile tiger puffer, Takifugu rubripes reared under different daily feeding protocols for 60 days. These parameters include
superoxide dismutase (SOD, A), catalase (CAT, B), glutathione (GSH, C) and glutathione peroxidase (GPx, D). P1, P2, P3 and P4 refer to groups fed for two daily
meals, four daily meals, six daily meals and continuous diurnal feeding, respectively. Values are expressed as the mean ± SD. Different letters indicate significant
differences between groups at P < 0.05.

Fig. 4. Temporal changes in the relative content (g dry matter per 100 g body weight) of the stomach in juvenile tiger puffer, Takifugu rubripes reared under different
feeding regimes. P1, P2, P3 and P4 refer to groups fed for two daily meals, four daily meals, six daily meals and continuous diurnal feeding, respectively. Points and
error bars represent the mean ± SD.

7
X.Q. Gao et al. Aquaculture 548 (2022) 737718

Fig. 5. Exponential curves showing gastric evacuation of digesta dry matter (g) per 100 g body weight over the 24 h sampling period for juvenile tiger puffer,
Takifugu rubripes reared under different feeding regimes. P1, P2, P3 and P4 refer to groups fed for two daily meals, four daily meals, six daily meals and continuous
diurnal feeding, respectively.

feed ingredients, feeding practices, and rearing conditions (Bonvini

Table 5
et al., 2018a; Bonvini et al., 2018b; Jafari et al., 2019; Busti et al., 2020).
The effect of feeding frequency on gastric evacuation rate (GER) and gastric
In the present study, feeding frequency did not affect serum TP, Alb, and
evacuation time (GET) of juvenile tiger puffer, Takifugu rubripes reared under
Glo contents, which suggests that the metabolic levels of plasma proteins
different feeding regimes for 60 days.
1
were not affected by feeding frequency. However, the serum Glu, TG,
Treatments GER (mg 100 g BW− GET Describing GER R2
and TC levels increased significantly with increasing feeding frequency,
min− 1) (min)
indicating that excessive food intake may induce enhanced accumula­
y = 6.3749e- tion of dietary carbohydrates and lipids in fish. A previous study
P1 5.12 187.69 0.307t 0.9123

y = 5.5822e-
P2 5.07 173.29 0.304t 0.9269
y = 6.3509e -
P3 4.98 190.32 0.299t 0.9251
y = 6.0329e-
P4 5.22 171.18 0.313t 0.9463
Note: GET represents the time necessary to empty half of the stomach contents.
P1, P2, P3 and P4 refer to groups fed for two daily meals, four daily meals, six
daily meals and continuous diurnal feeding, respectively.
studies (Gilannejad et al., 2017; Gilannejad et al., 2018). Evaluating the
dynamic changes in the digestive enzymes of this species is warranted in
future studies. However, studies have reported that high feeding fre­
quency led to better nutrient utilization in juvenile flounder (Para­
lichthys olivaceus) (Lee et al., 2000a), Australian snapper (Pagrus auratus)
(Tucker et al., 2006), and gilthead seabream (Sparus aurata L) (Andrew
et al., 2004). This inconsistency may be related to inter-species differ­
ences in stomach morphology. The above-mentioned species possess a Y-
shaped stomach, which allows newly ingested food to bypass acid
digestion. The bypassing effect can be stronger when feedings are more
frequent (Gilannejad et al., 2019). However, tiger puffer fish possess an
I-shaped stomach morphology with evacuation proceeding in sequential
order. For this species, shorter feeding intervals may induce fast passage
of food, thereby resulting in the loss of feed nutrients.
Hematological parameters can indicate the physiological status in
fish, and have been widely used to investigate the effects of different
observed that plasma cholesterol, triglycerides and glucose were
significantly lower in Jade Perch fed once a day compared with those fed
three times a day (Al-Khafaji et al., 2017). As in previous study, in our
research the significantly reduced serum levels of Glu, TG, and TC in the
P1 group, which indicates these fish most likely mobilized more energy
to maintain normal metabolic levels under starvation conditions.
Fish are often challenged by various stressors that originate from
environmental changes, including nutritional stress. Stress induces
antioxidant defense mechanisms in fish, triggering enhanced levels of
antioxidant enzymes to protect against the effects of reactive oxygen
species. According to the research findings, feeding could be one of the
biggest stressors for fish under farming conditions, since they are chal­
lenged with artificial diets and feeding strategies which are quite
different from that in their natural environment (López-Olmeda et al.,
2012). Hence, a wrong choice of feeding regime would increase its stress
levels (García and Villarroel, 2009). Several studies have characterized
that both low and high feeding frequencies could cause oxidative stress
and malondialdehyde accumulation (Li et al., 2014; Ding et al., 2017;
Guo et al., 2018). In the present study, the SOD, CAT, GSH, and GPx
activities of juvenile tiger puffer fish in the P1 and P4 groups were
significantly higher than those in the P2 and P3 groups, indicating that
low and high feeding frequencies might cause oxidative stress in juvenile
tiger puffer fish. These results support those of previous studies by Li
et al. (2014) and Guo et al. (2018), which demonstrate that inappro­
priate feeding can undoubtedly influence the health status of fish to a
certain extent. Tiger puffer fish are known to be fierce and cannibalistic.

8
X.Q. Gao et al.
It has been previously shown that the reduced food availability can
exacerbate aggression and cannibalistic behaviors among Channa stria­
tus juveniles (Qin and Fast, 1996a, 1996b). Our results suggest that due
to lack of food, juvenile tiger puffer fish in the P1 group exhibited higher
rates of cannibalism. Likewise, we observed that over-feeding in the P4
group led to intense competition for food intake, which increased the
chance of biting each other. Additionally, we also speculated that the
increased in antioxidant enzymes levels might be associated with the
altered physiological state of fish subjected to a heavy burden on the
digestive system under the condition of continuous diurnal feeding. In
all, the occurrence of these conditions may have caused oxidative stress,
but the exact mechanisms require further study.
This study is the first to report the gastric evacuation characteristics
of juvenile tiger puffer fish. In the present study, the relative stomach
contents of initial satiation in groups P1-P4 were basically the same, and
the average feed intake to apparent satiation at one meal was 3.63%,
which was consistent with the feeding rate set at the beginning of the
study. In addition, more than 50–60% of the meal emptied from the
stomach at postprandial 4–5 h, while the stomach fully emptied at
postprandial 10–12 h, suggesting that the stomach contents were
emptied quickly into the intestine and then digested. Gastric evacuation
rate (GER) experiments have been used to optimize feeding regimes for
farmed fish, helping to estimate stomach fullness at postprandial time
and return of appetite, as well as the body mass that provides optimum
food conversion efficiency and maximum growth rate (Lee et al., 2000b;
Bolliet et al., 2001; Dwyer et al., 2002; Riche et al., 2004; Booth et al.,
2008). The exponential model of GER to describe the gastric evacuation
of juvenile tiger puffer fish suggested a fast initial evacuation rate that
slows down as stomach fullness decreases (Nikolopoulou et al., 2011).
Similarly, the exponential model has been used to describe the gastric
evacuation or evacuation rates of several other carnivorous fish,
including gilthead sea bream, European sea bass (Dicentrarchus labrax
L.), Atlantic cod (Gadus morhua L), and Australian snapper (Dos and
Jobling, 1992; Booth et al., 2008; Nikolopoulou et al., 2011). Addi­
tionally, GER has been described using square root, square and cubic
root curves, linear, and logistic models for other carnivorous fish (Berens
and Murie, 2008; Khan et al., 2016; Jia et al., 2020; Skm et al., 2020).
The use of various models suggests that differences in gastric evacuation
patterns are likely to be attributable to variations in diet, species,
feeding regime, and environmental factors. We also observed that
feeding frequency had no effect on GER, which concurred with previ­
ously reported observations by Riche et al. (2004) and Booth et al.
(2008). Some studies have reported that a wide variety of factors may
affect GET, including fish and meal size, species, water temperature, and
feeding strategy (Gwyther and Grove, 1981; Venou et al., 2003; Bern­
reuther et al., 2008; Adamidou et al., 2009; De et al., 2016; Skm et al.,
2020). In this study, GER based on a single feeding to apparent satiation
was approximately 5.11 ± 0.09 g per 100 g BW per min, taking
approximately 180.62 ± 9.78 min (approximately 3 h) to digest 50% of
the ingested food in the stomach. Thus, the results support that a four
times/day feeding strategy may better align with the digestive physio­
logical characteristics of juvenile tiger puffer fish.
5. Conclusion
The study findings indicate that a feeding frequency of 4–6 times/
day could greatly improve the growth performance and feed conversion
ratio of this species, but four daily feedings induced optimal results.
Inappropriate feeding strategies (too low or too high feeding fre­
quencies) not only produced poor growth and feed conversion ratios, but
altered the blood physiology and induced a stress response. In addition,
the variation in feeding frequency had no effect on the GER. Meanwhile,
according to the GET calculate, approximately 50% of a single meal was
passed within 180.62 ± 9.78 min (about 3 h), which further confirmed
that feeding intervals in four times/day may better align with the
digestive physiological characteristics of juvenile tiger puffer fish.
9
Aquaculture 548 (2022) 737718
Overall, these results indicate that the best growth performance and
physiological indicators can be achieved when fed at 4 times/day.
Data availability statement
The data that support the findings of this study are openly available
in “figshare” at https://figshare.com/s/6ad874213c6b92ccaa17, DOI:
https://doi.org/10.6084/m9.figshare.14955147
Declaration of Competing Interest
All authors have no any potential sources of conflict of interest.
Acknowledgments
This study was supported by the National Key R&D Program of China
(No. 2019YFD0900503), Central Public-interest Scientific Institution
Basal Research Fund, YSFRI, CAFS (No. 20603022021007), Central
Public-interest Scientific Institution Basal Research Fund, CAFS (No.
2020TD49), and the Agriculture Research System of MOF and MARA
(CARS-47). We thank the Dalian Tianzheng Industrial Co. Ltd. for
providing the samples used in this study and the Yellow Sea Fisheries
Research Institute for their excellent technical assistance.
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