Aquaculture Reports 24 (2022) 101095

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

Response of lipid-related composition of farmed tiger puffer (Takifugu

rubripes) to starvation under different dietary lipid levels in the previous
feeding period
Zhangbin Liao a, b, Qingli Gong a, Yan Liu a, *, Yuliang Wei b, c, Mengqing Liang b, c,
Houguo Xu b, c, **
a
Fisheries College, Ocean University of China, 5 Yushan Road, Qingdao 266003, China
b
Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, 106 Nanjing Road, Qingdao 266071, China
c
Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, 1 Wenhai Road, Qingdao
266237, China

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

Keywords: Starvation is a natural process in the life cycle of wild fish, and also a means of body composition regulation for
Takifugu rubripes farmed fish. This study with tiger puffer was aimed at investigating the effects of starvation on lipid and fatty
Starvation acid compositions in fish with different levels of lipid accumulation. Different lipid accumulation levels in tiger
Lipid level
puffer were obtained by manipulating the dietary lipid content (8%, 12%, and 16%) in a previous feeding period
Fish quality
Fatty acid composition
for 9 weeks. Each diet was fed to sextuplicate net cages in this feeding trial. At the end of the feeding trial, fish
with an average initial body weight of 68.26 g were then subjected to starvation for 31 days. Tissue samples were
collected at the end of the feeding trial and at 1, 4, 9, 16, and 31 days after starvation. The results showed that
the muscle lipid content decreased linearly with starvation time, but the liver lipid content increased firstly (1–16
days of starvation), and thereafter sharply declined. However, no interaction between starvation time and dietary
lipid level was observed in the effects on liver and muscle lipid composition. Starvation had slight effects on
muscle and liver fatty acid compositions, except that it largely increased the C22:5n-3 content in the muscle.
Starvation primarily mobilized C14:0, C16:1n-7, C20:1n-9 and C22:1n-9 in the muscle, and C18:0, C18:1n-9, and
EPA in the liver. Interactive effects between starvation time and dietary lipid level were observed for many fatty
acids. Short-term (1–4 days) starvation increased the muscle texture parameters, but longer-term starvation
decreased them. In conclusion, starvation exerts significant effects on lipid and fatty acid composition in farmed
tiger puffer. Customed lipid and fatty acid composition, and possibly customed fillet quality could be obtained
through manipulation of starvation time and dietary lipid level in the previous feeding period.

1. Introduction improved the muscle color stability in Pacific bluefin tuna (Thunnus
orientalis) (Miyake et al., 2011), and increased the slaughter yield and
Feed restriction is a natural part of the life cycle of many wild fish produced a superior body shape in Atlantic salmon (Salmo salar) (Einen
populations and also applied in farmed fish (Davis and Gaylord, 2011). It et al., 1998).
has beneficial effects on not only stress resistance but also fillet quality Fish are the main source of long-chain polyunsaturated fatty acids
of farmed fish (HSA, 2005). In aquaculture species, feed deprivation has (LC-PUFA) for human consumption, and various measures have been
been demonstrated to be able to improve the fillet quality in gilthead sea taken to manipulate the lipid and fatty acid composition of farmed fish,
bream (Sparus aurata) (Ginés et al., 2002; Grigorakis and Alexis, 2005), among which starvation is a most important one (Ginés et al., 2002;
Atlantic halibut (Hippoglossus hippoglossus) (Foss et al., 2009), and Arctic Yildirim-Aksoy et al., 2010; Miyake et al., 2011). Besides starvation,
charr (Salvelinus alpinus) (Imsland et al., 2020). Starvation even dietary lipids, as the main source of energy provision and storage in fish

* Corresponding author.
** Corresponding author at: Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, 106 Nanjing Road, Qingdao 266071, China.
E-mail addresses: [email protected] (Y. Liu), [email protected] (H. Xu).

https://doi.org/10.1016/j.aqrep.2022.101095
Received 12 November 2021; Received in revised form 16 February 2022; Accepted 13 March 2022
Available online 24 March 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/).
Z. Liao et al. Aquaculture Reports 24 (2022) 101095

body (Tocher, 2003), also largely affect the lipid and fatty acid 8.05% (of dry matter), 12.02% and 16.36%, respectively. The diets were
composition of farmed fish. In particular, high dietary lipid levels are made following the standard procedures in our laboratory and stored at
being increasingly used in aquafeeds, in consideration of the − 20 ◦ C before use. The fatty acid composition of the experimental diets
protein-sparing effects of lipids (Cho and Kaushik, 1990; Ding et al., are presented in Table 2.
2010) and other specific demands for lipids like the
pigmentation-regulation demands (Torissen, 1985; Choubert et al., 2.2. Experimental fish, feeding procedure and sampling
1991; Mishra and Samantaray, 2004; Borges et al., 2009; Gao et al.,
2011; Arenas et al., 2021). In consequence, the application of high-lipid Juvenile tiger puffer (average initial body weight, 19.5 g) used in the
diets substantially affects the lipid and fatty acid accumulation in feeding trial were purchased from Haidu Aquaculture Co., Ltd. (Tang­
farmed fish, which could further affects the regulatory effects of star­ shan, Hebei Province, China), and the feeding trial was conduct there.
vation on lipid-related composition in farmed fish. Before the start of the feeding trial, experimental fish were reared in
Tiger puffer (Takifugu rubripes) is an important aquaculture species in indoor cement tanks (6.2 m × 6.2 m × 1.8 m) and fed a commercial feed
Asia, and also becoming a model fish for lipid studies because of its for 14 days to acclimate to the experimental conditions. During the
special lipid storage pattern. This fish species has low muscle lipid acclimation period, the lower teeth of fish were cut away with the nail
content and store lipids predominantly in the liver (Bjerkeng et al., clipper to prevent cannibalism. At the onset of the feeding trial, exper­
1997; Einen and Skrede, 1998). There used to be strict regulation on the imental fish were randomly distributed into 18 net cages (1.4 m × 1.4 m
marketing of this fish species because of its high tetrodotoxin contents. × 1.0 m) placed in the cement tanks. Placing net cages in a large water
However, in farmed tiger puffer, the tetrodotoxin content has been very body was beneficial to keeping a low water temperature that the fish
low or even non-detectable, promoting the marketing of this fish species, need. Each diet was randomly assigned to sextuplicate net cages, and
and also making the liver of this fish an edible part. It has been each net cage was stocked with 50 fish. Fish were hand-fed to apparent
demonstrated that the fillet quality of tiger puffer is sensitive to lipid satiation twice daily (6:00 and 18:00). Eighty percent of rearing water
composition (Einen and Skrede, 1998). In these considerations, the was changed after each feeding. The feeding period lasted 9 weeks (see
present study was aimed at investigating the efficacy of using starvation growth performance in Supplementary Table S1), after that, 30 fish of
as a means of lipid composition regulation in tiger puffer. This work was each net cage were randomly selected for a following starvation
conducted based on fish enriched with different lipid levels in a previous experiment. In the following starvation period, samples were collected
feeding trial, considering that various dietary lipid levels are being used at 5 sampling time points, namely, day 1, 4, 9, 16, and 31 (D1, D4, D9,
in tiger puffer farming, which subsequently result in various lipid D16, and D31) after starvation, respectively [the end of the feeding
accumulation levels in fish. Results of this study could add knowledge to period was designated as day 0 (D0)]. During the whole experiment, the
the fish product quality regulation through feeding strategies. water temperature ranged from 20◦ to 23◦ C; salinity, 22–30; pH,
7.4–8.2; and dissolved oxygen, 5–7 mg L− 1. No mortality was observed
2. Materials and methods during the starvation period.
Before sampling, fish were anesthetized with eugenol (1:10000).
2.1. Experimental diets in the previous feeding period Three randomly selected whole fish were collected from each net cage
for the analysis of proximate composition after D0, D16 and D31. Four
Three experimental diets were formulated (Table 1). A diet with a more randomly selected fish per net cage were dissected to collect the
suitable lipid content (8.0%) for tiger puffer was used as the control diet samples of liver and muscle for other assays at the 6 sampling time
(diet Control) (Takii et al., 1995; Kikuchi et al., 2009). The other two points. After dissection, four big pieces of muscle tissue (dorsal muscle,
diets with a moderately (12.0% of dry matter, diet MHL) or extremely about 3 cm × 1.5 cm), and the liver remnant (excluding the small tip
high lipid level (16.0%, diet EHL) were formulated by adding more fish part sampled) were collected for subsequent potential use. All tissue
oil into diet Control. The analyzed lipid level in these three diets was
Table 2
Table 1 Fatty acid composition of the experimental diets (% total fatty acids).
Formulation and proximate composition of the experiment diets (% dry matter
Fatty acids Control MHL EHL
basis).
C14:0 4.22 4.77 4.94
Ingredients Control MHL EHL
C16:0 11.34 11.59 11.47
Fish meal 40.0 40.0 40.0 C18:0 3.87 3.79 3.71
Soybean meal 8.00 8.00 8.00 C20:0 0.33 0.37 0.39
Soybean protein concentrate 6.00 6.00 6.00 C22:0 0.19 0.18 0.18
∑
Beer yeast 6.00 6.00 6.00 SFA1 19.95 20.70 20.70
Wheat meal 18.48 18.48 18.48 C16:1n-7 7.73 8.44 8.68
Casein 6.00 6.00 6.00 C18:1n-9 9.55 9.92 10.11
Vitamin premixa 0.20 0.20 0.20 C20:1n-9 3.17 4.10 4.76
Mineral premixa 0.50 0.50 0.50 C22:1n-9 0.49 0.62 0.71
Monocalcium phosphate 1.00 1.00 1.00 C24:1n-9 0.79 0.84 0.83
∑
Vitamin C 0.20 0.20 0.20 MUFA2 21.73 23.92 25.10
Choline chloride 0.20 0.20 0.20 C18:2n-6 20.55 17.62 16.23
Attracrant 0.30 0.30 0.30 C18:3n-6 0.15 0.16 0.16
Ethoxyquin 0.02 0.02 0.02 C20:2n-6 0.36 0.40 0.42
Mold inhibitor 0.10 0.10 0.10 C20:3n-6 0.15 0.15 0.17
Lecithin 1.00 1.00 1.00 C20:4n-6 1.10 1.18 1.21
∑
Fish oil 4.00 8.00 12.0 n-6PUFA3 22.32 19.52 18.19
Starch 8.00 4.00 0.00 C18:3n-3 3.07 3.06 3.10
Proximate composition C20:5n-3 11.96 11.96 11.91
Moisture 8.33 7.90 6.54 C22:6n-3 20.98 20.83 21.01
∑
Crude protein 50.62 50.83 51.13 n-3PUFA4 36.01 35.86 36.02
∑ ∑
Crude lipid 8.05 12.02 16.36 n-3/ n-6 1.61 1.84 1.98
Ash 9.58 9.64 9.67
Notes: 1SFA, saturated fatty acids; 2MUFA, mono-unsaturated fatty acids; 3n-
a
Vitamin premix and mineral premix, designed for marine fish, were pur­ 6PUFA, n-6 poly-unsaturated fatty acids; 4n-3 PUFA, n-3 poly-unsaturated fatty
chased from Qingdao Master Biotech Co., Ltd, Qingdao, China. acid.

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Z. Liao et al. Aquaculture Reports 24 (2022) 101095

samples were frozen with liquid nitrogen immediately, and then stored 3. Results
at − 76 ◦ C before use. All sampling protocols, as well as fish handing
practices, were reviewed and approved by the Animal Care and Use 3.1. Muscle and liver proximate composition
Committee of Yellow Sea Fisheries Research Institute.
Starvation resulted in significantly (P < 0.05) lower muscle lipid
2.3. Analysis of the proximate composition, fatty acid composition, and content, which in D31 was only nearly half that at D0 (Table 3). With
biochemical parameters of tissues progressing of starvation, the protein content first slightly decreased
(Day 0–16 of starvation) and then sharply decreased (D31), whereas the
The proximate composition analysis of experimental diets (sextu­ moisture content followed an opposite trend. In the liver, starvation
plicate assays for each diet), whole fish (three individual fish per tank), firstly significantly (P < 0.05) increased the lipid content (Day 0–16 of
muscle and liver (four samples per tank) was performed according to the starvation), and then sharply decreased it (Day 31), whereas the protein
standard methods of Association of Official Analytical Chemists. Briefly, and moisture contents showed opposite trends to the lipid content.
samples of diets and fish were oven-dried at 105 ◦ C to constant weight Expectedly, higher dietary lipid levels resulted in higher lipid con­
for moisture assay. Protein was determined by measuring nitrogen (N × tents in the two tissues. Significant (P < 0.01) interaction was observed
6.25) using the Kjeldahl method; lipid by petroleum ether extraction between effects of starvation and dietary lipid level on the muscle
using the Soxhlet method; and ash by incineration at 550 ◦ C. The lipid moisture and protein contents. The increase of muscle moisture by
content of the fish liver was assayed with the Soxhlet method, but the starvation, and decrease of muscle protein were more significant when
lipid in muscle was extracted and analyzed with the chloroform- fish were fed high-lipid diets. However, no such interaction was
methanol method according to Folch et al. (1957). observed in the liver.
The fatty acid compositions of diet (sextuplicate assays for each diet)
and fish tissues (a pooled muscle sample for each tank with three sam­ 3.2. Fatty acid composition in the muscle
ples from different fish) were analyzed with gas chromatograph (GC-
2010 Pro, Shimadzu, Japan). Briefly, fatty acids in freeze-dried samples Starvation resulted in slight change in muscle fatty acid composition
were saponified and methylated first with KOH-methanol and then with (Table 4). Contents of almost all the saturated fatty acids (SFA) and
HCL-methanol, in water bath at 72 ºC. Fatty acid methyl esters were monounsaturated fatty acids (MUFA) (except for C18:0 and C18:1n-9),
extracted with hexane and then subjected to the gas chromatography C18:3n-3, C20:3n-3, and C22:2n-6 in the muscle generally showed a
equipped with a fused silica capillary column (SH-RT-2560, 100 m × decreasing trend with starvation. C20:5n-3 (EPA) and C22:6n-3 (DHA)
0.25 mm × 0.20 µm, Shimadzu, Japan) and a flame ionization detector. were decreased only by long-term starvation (D31). In contrast, the
The column temperature was programmed to rise from150 ◦ C up to contents of C18:3n-6, C20:4n-6 (ARA) and C22:5n-3 (DPA) were
200 ◦ C at a rate of 15 ◦ C min− 1, and then from 200 ◦ C to 250 ◦ C at a rate increased by starvation. In particular, the muscle DPA content at D31
of 2 ◦ C min− 1. Both the injector and detector temperature were 250 ◦ C. was about 2.5 times that at D0. This elevation was more evident in fish
Results were expressed as the percentage of each fatty acid with respect fed MHL and EHL compared to fish fed the control diet.
to total fatty acids (TFA). Higher-lipid diets resulted in decreased contents of C16:0, C16:1n-7,
All the biochemical parameters of tissues, i.e., glycogen, total C18:1n-9, C18:2n-6, C20:2n-6, and EPA, but increased contents of
cholesterol (TC) and malondialdehyde (MDA) were analyzed using C20:0, C20:1n-9, C24:1n-9, ARA, and DHA.
commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, Significant (P < 0.05) interactions were observed between effects of
China) according to the manufacturer’s instructions. starvation and dietary lipid level on C20:1n-9, C18:3n-6, C20:2n-6,
C20:3n-6, C20:3n-3 and DPA in the muscle.
2.4. Analysis of texture profile
3.3. Fatty acid composition in the liver
The muscle samples were stored at − 26 ◦ C immediately after
collection. The texture of raw, skinned, and filleted muscle was assayed With progressing of starvation, marginal changes were observed in
after defrosted 6 months later. The texture profile Analysis (TPA) was the liver fatty acid composition (Table 5). Only the contents of C18:0,
performed with TMS-Pro Texture analyzer (Food Technology Corpora­ C18:1n-9, and EPA were slightly decreased by starvation. The liver EPA
tion, Virginia, USA) equipped with a 25 N load cell. The 8-mm cylin­ content was decreased in particular when fish were fed EHL and MHL.
drical compression detector was oriented per pendicular to the muscle, The liver C16:0 content was increased by long-term starvation (D31).
and the TPA measurements were carried out at ambient temperature Increasing dietary lipid levels increased the contents of C14:0,
(23 ◦ C). The compression rate was set to 30 mm min− 1; and 30% C20:1n-9, C22:1n-9, C24:1n-9, C20:3n-6, ARA, C18:3n-3, EPA and DHA,
deformation was selected to reduce the damage to muscle structures. Six but decreased the contents of C16:0, C18:0 and C18:2n-6.
mechanical texture parameters (hardness, adhesiveness, cohesiveness Significant (P < 0.05) interactions were observed between effects of
resilience, gumminess and chewiness) were calculated by the manu­ starvation and dietary lipid level on C16:0, C20:0, C22:0, C20:1n-9,
facture’s instruments. C22:1n-9, C18:3n-6, C20:3n-6, ARA, C22:2n-6 and EPA.

2.5. Statistical methods 3.4. Biochemical parameters in the fish tissues

All percentage data were arcsine transformed before analysis. The With progressing of starvation, the glycogen content in both muscle
data were analyzed by factorial (two-way) ANOVA. When a significant and liver was decreased rapidly from D0 to D4, and remained low until
main effect of factor 1 was observed, data were analyzed by one-way the end of the experiment (Table 6). Starvation firstly (D0-D16 and D0-
ANOVA followed by Tukey’s test to inspect all differences among the D9, respectively) increased the liver MDA and TC contents, and there­
treatments with or without factor 2. When a significant interaction was after decreased them. A synergetic increase of liver MDA and TC con­
observed, data were analyzed by one-way ANOVA followed by Tukey’s tents by dietary lipid level and starvation was observed.
test to inspect all differences among the dietary treatments and not ac­
cording to each main effect because of the interaction. Differences were 3.5. Muscle texture
regarded as significant when P < 0.05. All data are presented as means
± standard error and all statistical analyses were performed using SPSS Starvation exerted significant effects on the muscle texture param­
26.0 (SPSS Inc, USA). eters (Table 7). All the texture parameters reached plateau in value at D1

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Z. Liao et al. Aquaculture Reports 24 (2022) 101095

Table 3
Two-way ANOVA for effects of starvation and dietary lipid level on liver and muscle proximate composition of experimental tiger puffer (% wet weight, mean ±
standard error).
Variable Muscle Liver

Crude lipid Crude protein Moisture Crude lipid Crude protein Moisture

Individual treatment
Lipid level (%) Starvation day
8 0 0.83 ± 0.11 17.8 ± 0.14a 80.3 ± 0.16d 51.7 ± 0.48 6.81 ± 0.14 37.0 ± 1.54
8 1 0.67 ± 0.04 17.7 ± 0.28a 80.4 ± 0.30d 52.7 ± 1.44 6.41 ± 0.27 37.1 ± 1.68
8 4 0.68 ± 0.05 17.1 ± 0.26ab 81.2 ± 0.31cd 56.3 ± 0.64 6.83 ± 0.06 35.7 ± 1.47
8 9 0.64 ± 0.02 17.8 ± 0.53a 80.6 ± 0.40d 62.1 ± 1.24 6.22 ± 0.25 30.9 ± 0.98
8 16 0.62 ± 0.10 16.7 ± 0.27ab 81.8 ± 0.28cd 61.4 ± 1.02 6.09 ± 0.26 30.9 ± 0.83
8 31 0.52 ± 0.02 14.9 ± 0.20cd 83.8 ± 0.30b 46.9 ± 1.74 7.22 ± 0.48 45.1 ± 1.49
12 0 0.92 ± 0.08 17.3 ± 0.39ab 81.0 ± 0.35cd 53.0 ± 0.36 6.21 ± 0.12 36.7 ± 1.74
12 1 0.81 ± 0.05 17.5 ± 0.20ab 80.8 ± 0.19d 54.1 ± 1.36 6.95 ± 0.14 33.2 ± 1.08
12 4 0.75 ± 0.03 16.6 ± 0.27ab 81.8 ± 0.35cd 59.3 ± 2.41 6.39 ± 0.37 33.2 ± 2.49
12 9 0.73 ± 0.07 16.1 ± 0.38bc 82.4 ± 0.38bc 62.6 ± 2.24 6.30 ± 0.34 27.9 ± 1.37
12 16 0.69 ± 0.06 16.8 ± 0.29ab 81.8 ± 0.34cd 65.0 ± 1.43 6.05 ± 0.21 27.4 ± 1.19
12 31 0.57 ± 0.00 14.3 ± 0.37de 83.7 ± 0.30b 48.6 ± 1.62 7.46 ± 0.46 43.0 ± 1.43
16 0 1.25 ± 0.15 17.3 ± 0.40ab 81.1 ± 0.42cd 57.2 ± 1.45 6.22 ± 0.20 31.6 ± 0.85
16 1 0.83 ± 0.07 17.3 ± 0.16ab 80.8 ± 0.12d 59.1 ± 1.25 6.06 ± 0.14 32.4 ± 0.89
16 4 0.78 ± 0.07 16.7 ± 0.20ab 81.8 ± 0.19cd 62.3 ± 1.68 6.20 ± 0.17 29.0 ± 1.04
16 9 0.80 ± 0.06 16.8 ± 0.22ab 81.2 ± 0.23cd 66.6 ± 0.69 6.13 ± 0.27 26.5 ± 0.89
16 16 0.68 ± 0.03 17.0 ± 0.18ab 81.5 ± 0.16cd 69.1 ± 1.08 6.13 ± 0.28 24.3 ± 1.03
16 31 0.57 ± 0.05 12.7 ± 0.32e 86.1 ± 0.41a 51.7 ± 1.72 6.94 ± 0.03 40.3 ± 1.63
Pooled SE (One-way ANOVA) 0.023 0.153 0.178 0.700 0.072 0.694
Means of main effect
Lipid level (%) Starvation day
0 1.01 ± 0.08A 17.5 ± 0.19A 80.8 ± 0.20C 54.0 ± 0.75C 6.42 ± 0.11B 35.1 ± 0.98B
1 0.77 ± 0.03B 17.5 ± 0.13A 80.7 ± 0.13C 55.3 ± 0.99C 6.44 ± 0.14B 34.3 ± 0.90B
4 0.73 ± 0.03B 16.8 ± 0.14A 81.6 ± 0.17B 59.3 ± 1.14B 6.46 ± 0.16B 32.6 ± 1.18B
9 0.73 ± 0.04B 16.9 ± 0.32A 81.3 ± 0.27BC 63.9 ± 0.99A 6.22 ± 0.15B 28.5 ± 0.77C
16 0.67 ± 0.04BC 16.8 ± 0.13A 81.7 ± 0.14B 65.2 ± 0.99A 6.09 ± 0.14B 27.5 ± 0.95C
31 0.55 ± 0.02C 14.0 ± 0.29B 84.6 ± 0.42A 49.2 ± 1.06D 7.22 ± 0.22A 42.8 ± 0.97A
8 0.66 ± 0.03Y 17.0 ± 0.22X 81.2 ± 0.23Y 55.2 ± 1.04Y 6.59 ± 0.12 36.4 ± 1.02X
12 0.76 ± 0.03XY 16.4 ± 0.26Y 81.8 ± 0.22X 57.3 ± 1.17Y 6.56 ± 0.15 34.1 ± 1.21Y
16 0.84 ± 0.05X 16.4 ± 0.31Y 81.8 ± 0.30X 61.0 ± 1.13X 6.27 ± 0.09 31.0 ± 1.03Z
P-values of two-way ANOVA
Starvation 0.001 * 0.000 * 0.000 * 0.000 * 0.000 * 0.000 *
Lipid level 0.000 * 0.000 * 0.000 * 0.000 * 0.088 0.000 *
Starvation × Lipid level 0.283 0.007 * 0.001 * 0.978 0.479 0.687

Data in a same column not sharing a same superscript letter were significantly different (P < 0.05).

of starvation, and thereafter decreased. Long-term starvation decreased Although the muscle protein content has been changed, it was sub­
the cohesiveness, springiness and chewiness of muscle. Significant in­ stantially changed only at 31 days of starvation. This indicates that the
teractions were observed between effects of starvation and dietary lipid muscle protein was mobilized as energy source at a low priority. In
level on hardness and gumminess. contrast, the glycogen was mobilized at 4 days of starvation. Protein is
stable component of fish body and less susceptible to feed factors
4. Discussion (Shearer, 1994). In Pacific bluefin tuna, the muscle protein content was
not influenced by 6 days of starvation (Miyake et al., 2011). Similarly,
The present results confirmed the significant regulation of fish body long-term starvation (more than 2 months) resulted in no significant
composition by starvation. Muscle lipid content largely affects the fillet change in the muscle protein level of marketable-size Atlantic salmon
quality (Einen, and Skrede, 1998; Ruiz-Carrascal, 2000, Rasmussen, (Einen and Thomassen, 1998).
2001; Johnston et al., 2006; Suárez et al., 2014), and is often the firstly Different from the present study, in a recent and very similar study
concerned composition trait in starvation studies. In this study, starva­ on turbot, which is also a lean fish species (Scophthalmus maximus), it
tion linearly decreased the muscle lipid content, indicating the mobili­ was observed that starvation increased the lipid content in the muscle
zation of lipid for energy purpose. The lipid content in muscle of tiger (Xu et al., 2022). In another lean species red seabream (Pagrus major),
puffer is lower (around 1% of wet weight) than other fish species, for the mucle lipid content was increased even by a very short-term (5 days)
example, much lower compared to typical fatty fish, salmonids, of which starvation (Kaneko et al., 2016). In a freshwater species grass carp
the lipid content can be as high as 15–20%. Therefore, the fillet quality (Ctenopharyngodon idellus), during overwintering an 8-week starvation
parameters, for example the texture, could be sensitive to lipid content. significantly increased the triglycerides content of muscle (Sun et al.,
Many previous studies have shown that the increase of lipid content 2021). The difference between results from tiger puffer and grass carp
leads to softening of fish flesh, namely, decrease in firmness, and vice was more easily explained considering that grass carp is herbivorous and
versa (Suárez et al., 2014). The concurrent change of muscle texture and glycogen metabolism may play more important roles in
lipid content by starvation was observed in the present study, which starvation-stimulated energy mobilization (Sun et al., 2021). However
showed that the cohesiveness, springiness and chewiness were signifi­ the difference among results from tiger puffer, turbot, and red seabream
cantly reduced by starvation, associated with the decreased lipid con­ was difficult to explain considering that all these three species are lean
tent. Nevertheless, the change of muscle texture could not be only species and are of similar trophic level. A general speculation was that
attributed to the change of lipid content, because the protein content tiger puffer store most of the lipid in liver and thus had the lowest muscle
was also reduced by starvation, and the moisture content was changed in lipid content among these three species. As mentioned previously, this
an opposite way. characteristic may make the muscle lipid more susceptible to energy

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Z. Liao et al. Aquaculture Reports 24 (2022) 101095

Table 4
Two-way ANOVA for effects of starvation and dietary lipid level on muscle fatty acid composition of experimental tiger puffer (% total fatty acids, mean ± standard
error).
Variable C14:0 C16:0 C18:0 C20:0 C22:0 SFA C16:1n-7 C18:1n-9 C20:1n-9

Individual treatment
Lipid level Starvation
(%) day
8 0 0.67 ± 0.05 23.3 ± 0.69 11.8 ± 0.21 0.44 ± 0.02 0.15 ± 0.01 36.4 ± 0.91 1.06 ± 0.05 11.5 ± 0.14 1.30 ±
0.02def
8 1 0.64 ± 0.03 23.3 ± 0.32 12.2 ± 0.13 0.38 ± 0.01 0.12 ± 0.00 36.7 ± 0.27 0.99 ± 0.03 11.9 ± 0.14 1.18 ± 0.03efg
8 4 0.54 ± 0.03 23.2 ± 0.28 12.4 ± 0.15 0.37 ± 0.01 0.10 ± 0.01 36.6 ± 0.34 0.87 ± 0.03 11.5 ± 0.18 1.05 ±
0.02fgh
8 9 0.41 ± 0.02 23.1 ± 0.12 13.4 ± 0.07 0.37 ± 0.01 0.10 ± 0.00 37.4 ± 0.10 0.79 ± 0.02 11.9 ± 0.21 1.01 ± 0.04gh
8 16 0.29 ± 0.02 21.7 ± 0.18 12.8 ± 0.39 0.33 ± 0.01 0.08 ± 0.00 35.3 ± 0.46 0.75 ± 0.04 11.7 ± 0.16 0.90 ± 0.02 h
8 31 0.33 ± 0.03 23.2 ± 0.37 12.6 ± 0.42 0.43 ± 0.01 0.12 ± 0.02 36.7 ± 0.08 1.13 ± 0.06 12.0 ± 1.09 1.37 ±
0.24cde
12 0 0.59 ± 0.04 22.2 ± 0.37 12.0 ± 0.28 0.51 ± 0.04 0.17 ± 0.04 35.5 ± 0.55 0.93 ± 0.04 11.3 ± 0.20 1.63 ±
0.06abc
12 1 0.64 ± 0.03 23.2 ± 0.21 12.2 ± 0.49 0.48 ± 0.02 0.12 ± 0.01 36.5 ± 0.69 0.93 ± 0.05 11.6 ± 0.16 1.64 ±
0.01abc
12 4 0.52 ± 0.01 23.0 ± 0.62 12.7 ± 0.32 0.42 ± 0.01 0.12 ± 0.01 36.8 ± 0.96 0.78 ± 0.01 11.3 ± 0.14 1.37 ±
0.04cde
12 9 0.40 ± 0.01 22.6 ± 0.22 13.5 ± 0.11 0.43 ± 0.01 0.11 ± 0.00 37.0 ± 0.25 0.77 ± 0.02 11.8 ± 0.22 1.25 ±
0.02defg
12 16 0.25 ± 0.06 21.2 ± 0.25 12.0 ± 0.40 0.35 ± 0.01 0.08 ± 0.00 33.9 ± 0.55 0.73 ± 0.02 11.5 ± 0.38 1.08 ±
0.03fgh
12 31 0.33 ± 0.01 21.7 ± 0.21 12.2 ± 0.17 0.43 ± 0.01 0.09 ± 0.00 34.8 ± 0.02 1.19 ± 0.05 11.1 ± 0.20 1.39 ±
0.02bcde
16 0 0.66 ± 0.05 22.6 ± 0.16 11.7 ± 0.45 0.47 ± 0.03 0.13 ± 0.01 35.6 ± 0.45 0.91 ± 0.05 11.0 ± 0.11 1.66 ± 0.08ab
16 1 0.63 ± 0.01 22.8 ± 0.19 12.3 ± 0.09 0.46 ± 0.01 0.14 ± 0.00 36.4 ± 0.24 0.83 ± 0.01 11.4 ± 0.12 1.66 ± 0.04a
16 4 0.50 ± 0.03 23.0 ± 0.64 12.4 ± 0.25 0.43 ± 0.01 0.12 ± 0.00 36.4 ± 0.57 0.75 ± 0.03 11.2 ± 0.10 1.43 ±
0.02abcde
16 9 0.42 ± 0.02 22.6 ± 0.36 13.3 ± 0.22 0.46 ± 0.02 0.12 ± 0.00 37.0 ± 0.51 0.73 ± 0.02 12.2 ± 0.15 1.39 ±
0.05bcde
16 16 0.35 ± 0.01 21.4 ± 0.32 12.5 ± 0.34 0.39 ± 0.02 0.11 ± 0.01 34.8 ± 0.55 0.73 ± 0.01 11.4 ± 0.20 1.21 ± 0.03efg
16 31 0.33 ± 0.04 20.1 ± 0.04 12.6 ± 0.85 0.47 ± 0.03 0.10 ± 0.02 33.5 ± 0.92 1.17 ± 0.07 11.3 ± 0.49 1.51 ±
0.05abcd
Pooled SE 0.016 0.119 0.086 0.007 0.004 0.162 0.017 0.061 0.028
Means of main effect
Lipid level Starvation
(%) day
0 0.64 ± 22.7 ± 11.9 ± 0.47 ± 0.15 ± 0.01A 35.8 ± 0.97 ± 11.3 ± 1.53 ± 0.05A
0.03A 0.27A 0.18B 0.02A 0.37AB 0.03B 0.10B
1 0.64 ± 23.1 ± 12.2 ± 0.44 ± 0.13 ± 36.5 ± 0.92 ± 11.6 ± 1.48 ± 0.07A
0.01A 0.15A 0.14B 0.01AB 0.00AB 0.22A 0.03B 0.09AB
4 0.52 ± 23.1 ± 12.5 ± 0.40 ± 0.11 ± 36.6 ± 0.81 ± 11.3 ± 1.27 ± 0.05B
0.01B 0.28A 0.14B 0.01BC 0.00BC 0.35A 0.02C 0.09B
9 0.41 ± 22.8 ± 13.4 ± 0.42 ± 0.11 ± 37.1 ± 0.76 ± 12.0 ± 1.21 ± 0.05B
0.01C 0.16A 0.08A 0.01AB 0.00BC 0.19A 0.01C 0.11A
16 0.30 ± 21.5 ± 12.5 ± 0.36 ± 0.09 ± 0.00C 34.7 ± 0.74 ± 11.5 ± 1.06 ± 0.04C
0.02D 0.15B 0.22B 0.01C 0.31B 0.01C 0.14AB
31 0.33 ± 21.6 ± 12.5 ± 0.44 ± 0.10 ± 35.0 ± 1.16 ± 11.5 ± 1.42 ± 0.07A
0.01CD 0.47B 0.28B 0.01AB 0.01BC 0.54B 0.03A 0.38AB
8 0.49 ± 0.03 22.9 ± 12.5 ± 0.13 0.38 ± 0.11 ± 0.01 36.5 ± 0.92 ± 11.7 ± 1.11 ± 0.04Z
0.18X 0.01Y 0.23X 0.03X 0.11X
12 0.46 ± 0.03 22.3 ± 12.4 ± 0.16 0.44 ± 0.12 ± 0.01 35.7 ± 0.87 ± 11.4 ± 1.40 ± 0.04Y
0.20Y 0.01X 0.33Y 0.03XY 0.10Y
16 0.49 ± 0.03 22.2 ± 12.4 ± 0.16 0.44 ± 0.12 ± 0.00 35.7 ± 0.83 ± 11.4 ± 1.47 ± 0.04X
0.22Y 0.01X 0.28Y 0.03Y 0.10Y
P-values of two-way ANOVA
Starvation 0.000 * 0.000 * 0.000 * 0.000 * 0.000 * 0.000 * 0.000 * 0.008 * 0.000 *
Lipid level 0.348 0.000 * 0.819 0.000 * 0.686 0.017 * 0.001 * 0.046 * 0.000 *
Starvation×Lipid level 0.686 0.112 0.852 0.379 0.218 0.485 0.204 0.781 0.048 *

Variable C22:1n-9 C24:1n-9 MUFA C18:2n-6 C18:3n-6 C20:2n-6 C20:3n-6 C20:4n-6 C22:2n-6
Table 4 (-continued) Two-way ANOVA for effects of starvation and dietary lipid level on muscle fatty acid composition of experimental tiger puffer (% total fatty acids, mean ±
standard error)
Individual treatment
Lipid level Starvation
(%) day
8 0 0.30 ± 0.01 0.36 ± 0.02 14.0 ± 0.18 11.0 ± 0.16 0.07 ± 0.78 ± 0.18 ± 1.70 ± 0.03 0.14 ± 0.01
0.01bcd 0.03a 0.01a
8 1 0.27 ± 0.01 0.36 ± 0.02 14.7 ± 0.14 11.0 ± 0.24 0.06 ± 0.73 ± 0.13 ± 1.75 ± 0.03 0.09 ± 0.01
0.01bcd 0.02ab 0.01b
8 4 0.22 ± 0.01 0.27 ± 0.02 13.9 ± 0.15 10.8 ± 0.21 0.06 ± 0.69 ± 0.13 ± 1.75 ± 0.03 0.08 ± 0.01
0.01bcd 0.02abc 0.00b
(continued on next page)

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Z. Liao et al. Aquaculture Reports 24 (2022) 101095

Table 4 (continued )
Variable C14:0 C16:0 C18:0 C20:0 C22:0 SFA C16:1n-7 C18:1n-9 C20:1n-9

8 9 0.21 ± 0.01 0.31 ± 0.00 14.3 ± 0.25 10.4 ± 0.15 0.07 ± 0.68 ± 0.12 ± 1.87 ± 0.02 0.05 ± 0.00
0.00abcd 0.02abc 0.00b
8 16 0.25 ± 0.01 0.30 ± 0.01 13.9 ± 0.17 10.6 ± 0.15 0.06 ± 0.60 ± 0.12 ± 1.90 ± 0.03 0.05 ± 0.00
0.01bcd 0.01cd 0.00b
8 31 0.23 ± 0.06 0.44 ± 0.09 15.2 ± 1.53 10.6 ± 0.39 0.07 ± 0.63 ± 0.13 ± 1.92 ± 0.29 0.06 ± 0.01
0.00abcd 0.04bcd 0.01b
12 0 0.35 ± 0.04 0.45 ± 0.05 14.7 ± 0.27 9.31 ± 0.23 0.05 ± 0.64 ± 0.14 ± 1.88 ± 0.07 0.12 ± 0.03
0.01cd 0.01bcd 0.00b
12 1 0.34 ± 0.02 0.41 ± 0.01 14.9 ± 0.17 9.22 ± 0.14 0.06 ± 0.64 ± 0.14 ± 1.89 ± 0.03 0.08 ± 0.00
0.02bcd 0.01bcd 0.01b
12 4 0.27 ± 0.02 0.32 ± 0.01 14.0 ± 0.19 9.15 ± 0.14 0.10 ± 0.62 ± 0.13 ± 1.95 ± 0.03 0.07 ± 0.00
0.01ab 0.02bcd 0.00b
12 9 0.24 ± 0.01 0.39 ± 0.02 14.5 ± 0.25 9.21 ± 0.20 0.09 ± 0.62 ± 0.13 ± 2.24 ± 0.06 0.06 ± 0.00
0.00abc 0.00cd 0.01b
12 16 0.23 ± 0.03 0.31 ± 0.02 13.8 ± 0.43 8.99 ± 0.29 0.10 ± 0.55 ± 0.13 ± 2.22 ± 0.03 0.08 ± 0.03
0.01ab 0.02d 0.00b
12 31 0.18 ± 0.01 0.46 ± 0.01 14.3 ± 0.22 9.74 ± 0.42 0.12 ± 0.01a 0.64 ± 0.15 ± 2.27 ± 0.04 0.06 ± 0.00
0.02bcd 0.00b
16 0 0.28 ± 0.02 0.43 ± 0.03 14.3 ± 0.19 8.41 ± 0.13 0.05 ± 0.59 ± 0.14 ± 2.12 ± 0.06 0.07 ± 0.00
0.01bcd 0.01cd 0.00b
16 1 0.33 ± 0.01 0.47 ± 0.02 14.7 ± 0.13 8.17 ± 0.19 0.03 ± 0.00d 0.59 ± 0.13 ± 2.11 ± 0.03 0.08 ± 0.00
0.01cd 0.00b
16 4 0.29 ± 0.02 0.34 ± 0.01 14.0 ± 0.11 7.91 ± 0.26 0.09 ± 0.54 ± 0.14 ± 2.18 ± 0.03 0.06 ± 0.01
0.00abc 0.01d 0.00b
16 9 0.25 ± 0.01 0.43 ± 0.02 15.0 ± 0.11 8.09 ± 0.26 0.07 ± 0.64 ± 0.13 ± 2.40 ± 0.05 0.07 ± 0.01
0.01abcd 0.05bcd 0.01b
16 16 0.24 ± 0.02 0.40 ± 0.04 14.0 ± 0.24 8.17 ± 0.23 0.10 ± 0.53 ± 0.15 ± 2.51 ± 0.03 0.05 ± 0.01
0.00ab 0.01d 0.00b
16 31 0.20 ± 0.01 0.46 ± 0.06 14.7 ± 0.61 8.05 ± 0.46 0.10 ± 0.62 ± 0.14 ± 2.68 ± 0.30 0.07 ± 0.01
0.01ab 0.04cd 0.01b
Pooled SE 0.007 0.009 0.078 0.128 0.003 0.008 0.002 0.032 0.006
Means of main effect
Lipid level Starvation
(%) day
0 0.31 ± 0.41 ± 14.5 ± 9.57 ± 0.28 0.06 ± 0.01B 0.67 ± 0.15 ± 1.90 ± 0.12 ± 0.01A
0.02A 0.02AB 0.12AB 0.02A 0.01A 0.05B
1 0.31 ± 0.41 ± 14.8 ± 9.46 ± 0.35 0.05 ± 0.01B 0.65 ± 0.13 ± 1.92 ± 0.10 ± 0.02AB
0.01A 0.02AB 0.08A 0.02A 0.00B 0.04B
4 0.26 ± 0.31 ± 14.0 ± 9.38 ± 0.33 0.08 ± 0.62 ± 0.13 ± 1.95 ± 0.07 ± 0.00AB
0.01B 0.01C 0.09B 0.01AB 0.02A 0.00B 0.05B
9 0.23 ± 0.38 ± 14.6 ± 9.25 ± 0.30 0.08 ± 0.65 ± 0.13 ± 2.17 ± 0.06 ± 0.00B
0.01BC 0.02BC 0.14AB 0.01AB 0.02A 0.00B 0.07A
16 0.24 ± 0.34 ± 13.9 ± 9.26 ± 0.28 0.09 ± 0.01A 0.56 ± 0.13 ± 2.21 ± 0.06 ± 0.01B
0.01BC 0.02C 0.15B 0.01B 0.00B 0.07A
31 0.20 ± 0.45 ± 14.7 ± 9.48 ± 0.43 0.10 ± 0.01A 0.63 ± 0.14 ± 2.29 ± 0.07 ± 0.00B
0.02C 0.03A 0.50A 0.02A 0.01B 0.16A
8 0.25 ± 0.01 0.33 ± 14.3 ± 0.16 10.8 ± 0.07 ± 0.00Y 0.69 ± 0.14 ± 0.01 1.81 ± 0.08 ± 0.01
0.01Y 0.08X 0.01X 0.03Z
X
12 0.28 ± 0.02 0.39 ± 14.4 ± 0.13 9.24 ± 0.09 ± 0.01 0.62 ± 0.14 ± 0.00 2.05 ± 0.09 ± 0.01
0.02X 0.10Y 0.01Y 0.04Y
16 0.27 ± 0.01 0.42 ± 14.4 ± 0.11 8.15 ± 0.08 ± 0.58 ± 0.14 ± 0.00 2.31 ± 0.07 ± 0.00
0.01X 0.09Z 0.01XY 0.01Z 0.05X
P-values of two-way ANOVA
Starvation 0.000 * 0.000 * 0.000 * 0.322 0.000 * 0.000 * 0.000 * 0.000 * 0.004 *
Lipid level 0.167 0.000 * 0.907 0.000 * 0.003 * 0.000 * 0.819 0.000 * 0.306
Starvation × Lipid level 0.063 * 0.681 0.699 0.755 0.002 * 0.002 * 0.000 * 0.326 0.514

Variable n-6PUFA C18:3n-3 C20:3n-3 C20:5n-3 C22:5n-3 C22:6n-3 n-3PUFA n-3/n-6

Table 4 (-continued) Two-way ANOVA for effects of starvation and dietary lipid level on muscle fatty acid composition of experimental tiger puffer (% total fatty
acids, mean ± standard error)
Individual treatment
Lipid level Starvation
(%) day
8 0 13.8 ± 0.15 0.52 ± 0.03 0.25 ± 5.58 ± 0.18 4.07 ± 0.09ef 24.9 ± 0.91 35.3 ± 1.14 2.55 ± 0.10
0.02a
8 1 13.7 ± 0.24 0.42 ± 0.01 0.18 ± 5.41 ± 0.10 4.39 ± 0.32ef 24.5 ± 0.44 34.9 ± 0.57 2.55 ± 0.09
0.01abc
ef
8 4 13.5 ± 0.18 0.43 ± 0.04 0.17 ± 5.45 ± 0.11 4.24 ± 0.10 25.8 ± 0.46 36.1 ± 0.59 2.68 ± 0.07
0.00bc
8 9 13.2 ± 0.14 0.32 ± 0.01 0.14 ± 5.48 ± 0.06 3.90 ± 0.04ef 25.3 ± 0.32 35.1 ± 0.30 2.66 ± 0.04
0.01bc
8 16 13.3 ± 0.16 0.30 ± 0.02 0.14 ± 5.48 ± 0.11 6.07 ± 25.6 ± 0.36 37.6 ± 0.59 2.83 ± 0.07
0.01bc 0.24cde
8 31 13.4 ± 0.52 0.37 ± 0.09 0.13 ± 4.71 ± 0.57 6.04 ± 23.4 ± 0.31 34.7 ± 1.04 2.59 ± 0.05
0.01bc 0.67cde
(continued on next page)

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Z. Liao et al. Aquaculture Reports 24 (2022) 101095

Table 4 (continued )
Variable C14:0 C16:0 C18:0 C20:0 C22:0 SFA C16:1n-7 C18:1n-9 C20:1n-9

12 0 12.1 ± 0.26 0.54 ± 0.08 0.20 ± 5.41 ± 0.13 3.79 ± 0.08ef 27.8 ± 0.58 37.7 ± 0.69 3.11 ± 0.09
0.01ab
12 1 12.1 ± 0.12 0.37 ± 0.03 0.19 ± 5.43 ± 0.27 3.59 ± 0.11 f 26.9 ± 0.22 36.5 ± 0.54 3.03 ± 0.04
0.01abc
ef
12 4 12.0 ± 0.18 0.45 ± 0.03 0.15 ± 5.15 ± 0.18 4.00 ± 0.19 27.5 ± 1.03 37.2 ± 1.15 3.11 ± 0.11
0.01bc
12 9 12.3 ± 0.20 0.29 ± 0.01 0.15 ± 5.20 ± 0.08 3.51 ± 0.02 f 27.1 ± 0.28 36.3 ± 0.35 2.96 ± 0.07
0.01bc
12 16 12.1 ± 0.28 0.30 ± 0.01 0.13 ± 5.35 ± 0.18 7.02 ± 27.4 ± 0.89 40.2 ± 0.99 3.35 ± 0.15
0.01bc 0.74bc
12 31 12.9 ± 0.43 0.36 ± 0.06 0.14 ± 4.57 ± 0.05 8.74 ± 24.2 ± 0.18 38.0 ± 0.67 2.95 ± 0.15
0.00bc 0.68ab
16 0 11.3 ± 0.13 0.44 ± 0.03 0.16 ± 5.34 ± 0.15 3.52 ± 0.09 f 29.3 ± 0.54 38.8 ± 0.61 3.42 ± 0.07
0.02bc
16 1 11.1 ± 0.19 0.36 ± 0.00 0.18 ± 5.11 ± 0.05 3.28 ± 0.07 f 28.9 ± 0.40 37.8 ± 0.35 3.42 ± 0.08
0.01abc
16 4 10.9 ± 0.24 0.37 ± 0.05 0.14 ± 5.13 ± 0.20 4.64 ± 28.5 ± 0.55 38.7 ± 0.33 3.57 ± 0.06
0.02bc 0.42def
16 9 11.4 ± 0.20 0.29 ± 0.01 0.20 ± 5.18 ± 0.18 3.46 ± 0.13 f 27.6 ± 0.48 36.7 ± 0.59 3.24 ± 0.08
0.05ab
16 16 11.5 ± 0.23 0.29 ± 0.01 0.14 ± 5.14 ± 0.21 6.89 ± 27.4 ± 0.93 39.8 ± 0.81 3.48 ± 0.12
0.01bc 1.10bcd
16 31 11.6 ± 0.37 0.26 ± 0.03 0.11 ± 4.03 ± 0.31 9.77 ± 0.78a 26.1 ± 0.77 40.3 ± 1.51 3.48 ± 0.20
0.00c
Pooled SE 0.114 0.012 0.058 0.051 0.202 0.217 0.240 0.042
Means of main effect
Lipid level Starvation
(%) day
0 12.4 ± 0.27 0.50 ± 0.20 ± 5.44 ± 3.79 ± 0.07C 27.3 ± 37.3 ± 3.03 ±
0.03A 0.01A 0.09A 0.59A 0.58AB 0.10AB
1 12.3 ± 0.33 0.38 ± 0.18 ± 5.31 ± 3.77 ± 0.17C 26.8 ± 36.4 ± 3.00 ±
0.01BC 0.00AB 0.09A 0.55A 0.43B 0.11AB
4 12.2 ± 0.30 0.42 ± 0.16 ± 5.26 ± 4.29 ± 0.15C 27.2 ± 37.3 ± 3.09 ±
0.02AB 0.01BC 0.09A 0.47A 0.49AB 0.11AB
9 12.3 ± 0.24 0.30 ± 0.16 ± 5.29 ± 3.63 ± 0.07C 26.6 ± 36.0 ± 2.95 ±
0.01C 0.02BC 0.08A 0.35A 0.30B 0.08B
16 12.3 ± 0.23 0.30 ± 0.14 ± 5.32 ± 6.64 ± 0.44B 26.8 ± 39.1 ± 3.21 ±
0.01C 0.00C 0.10A 0.46A 0.52A 0.09A
31 12.6 ± 0.35 0.33 ± 0.13 ± 4.43 ± 8.18 ± 0.66A 24.6 ± 37.7 ± 3.01 ±
0.04BC 0.00C 0.22B 0.46B 0.99AB 0.15AB
8 13.5 ± 0.39 ± 0.17 ± 0.01 5.41 ± 4.71 ± 0.18 25.1 ± 35.7 ± 2.65 ±
0.09X 0.02X 0.08X 0.25Z 0.33Y 0.03Z
12 12.2 ± 0.40 ± 0.16 ± 0.01 5.23 ± 4.91 ± 0.39 27.0 ± 37.7 ± 3.10 ±
0.11Y 0.03X 0.08XY 0.33Y 0.41X 0.05Y
16 11.3 ± 0.34 ± 0.16 ± 0.01 5.07 ± 4.96 ± 0.45 28.1 ± 38.6 ± 3.44 ±
0.09Z 0.02Y 0.10Y 0.31X 0.33X 0.04X
P-values of two-way ANOVA
Starvation 0.180 0.000 * 0.000 * 0.000 * 0.000 * 0.000 * 0.000 * 0.009 *
Lipid level 0.000 * 0.041 * 0.332 0.003 * 0.176 0.000 * 0.000 * 0.000 *
Starvation × Lipid level 0.187 0.923 0.001 * 0.967 0.002 * 0.456 0.603 0.545

Data in a same column not sharing a same superscript letter were significantly different (P < 0.05). * : P < 0.05. SFA: saturated fatty acid.
Data in a same column not sharing a same superscript letter were significantly different (P < 0.05). * : P < 0.05. MUFA: monounsaturated fatty acid.
Data in a same column not sharing a same superscript letter were significantly different (P < 0.05). * : P < 0.05. PUFA: Polyunsaturated fatty acid.

mobilization. Both the studies on red seabream and grass carp suggested effects of short-term starvation on liver health. Decrease of liver lipid
that a lipoprotein lipase-mediated lipid transport mechanism could be content by longer-term starvation was probably due to the mobilization
involved in the increase of muscle lipid content by starvation (Kaneko for energy purpose. On the other hand, as expected, higher dietary lipid
et al., 2016; Sun et al., 2021). However, the corresponding precise levels resulted in higher liver lipid contents and lower moisture con­
mechanisms in turbot and tiger puffer remain unknown and warrant tents, as observed in other species such as grass carp (Du et al., 2005),
further studies. channel catfish (Ictalurus punctatus) (Yildirim-Aksoy et al, 2010) and
As mentioned previously, for farmed tiger puffer, which have very giant croaker (Nibea japonica) (Han et al., 2014), and also in our pre­
low or non-detectable levels of tetrodotoxin, liver has been an edible vious studies with tiger puffer (Xu et al., 2018). The non-response of
part and often is popular in some recipes because of its high lipid con­ liver protein content to starvation could be related to the lower priority
tent. Interestingly, the liver lipid content in the present study was in energy supply by protein compared to lipid (Lu et al., 2019).
significantly increased in the first 16 days of starvation and then Starvation resulted in slight change in muscle fatty acid composition,
decreased from 16 to 31 days of starvation. The lipid increase by short- but most fatty acids still showed a decreasing trend in response to
term starvation could be related to the metabolic disorder associated starvation. Importantly, only long-term starvation decreased the con­
with starvation. The fasting-caused fatty liver was observed in both mice tents of DHA and EPA in the muscle. This indicates that if short-term
and zebrafish (Danio rerio) (He et al., 2020; Xu et al., 2021). Tiger puffer starvation is used as a means of fillet quality regulation, the DHA and
already had a very high basic lipid content in the liver in normal con­ EPA contents, which are beneficial to health of human consumers, will
ditions. Therefore, when starvation is used as a means of body compo­ not be influenced. This facilitated the application of such strategies in
sition manipulation, careful attention should be paid on the harmful aquaculture practice. It is well known that DHA and EPA, which play

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Z. Liao et al. Aquaculture Reports 24 (2022) 101095

Table 5
Two-way ANOVA for effects of starvation and dietary lipid level on liver fatty acid composition of experimental tiger puffer (% total fatty acids, mean ± standard
error).
Variable C14:0 C16:0 C18:0 C20:0 C22:0 SFA C16:1n-7 C18:1n-9 C20:1n-9

Individual treatment
Lipid level Starvation
(%) day
8 0 3.32 ± 0.11 24.0 ± 9.55 ± 0.17 0.38 ± 0.02a 0.18 ± 37.4 ± 0.27a 8.02 ± 22.6 ± 0.70 3.22 ±
0.33abcd 0.01abc 0.19 0.14cde
8 1 3.23 ± 0.07 24.3 ± 8.72 ± 0.20 0.31 ± 0.15 ± 36.7 ± 7.84 ± 23.2 ± 0.48 2.42 ± 0.17f
0.32abc 0.01abc 0.01abc 0.25ab 0.18
8 4 3.34 ± 0.04 23.9 ± 8.84 ± 0.15 0.32 ± 0.15 ± 36.6 ± 8.17 ± 23.5 ± 0.40 2.88 ± 0.16ef
0.34bcd 0.01abc 0.00abc 0.46abc 0.08
8 9 3.30 ± 0.14 23.7 ± 8.71 ± 0.06 0.35 ± 0.18 ± 36.2 ± 8.41 ± 22.5 ± 0.22 3.20 ±
0.47bcd 0.02abc 0.00abc 0.57abcd 0.19 0.15cdef
8 16 3.26 ± 0.04 23.5 ± 8.16 ± 0.19 0.32 ± 0.16 ± 35.4 ± 8.56 ± 22.2 ± 0.55 2.95 ± 0.12ef
0.21bcd 0.02abc 0.01abc 0.26abcde 0.25
8 31 3.43 ± 0.14 24.3 ± 7.29 ± 0.49 0.32 ± 0.15 ± 35.5 ± 8.94 ± 21.5 ± 0.54 3.27 ±
0.88abc 0.01abc 0.02abc 1.21abcde 0.25 0.33bcde
12 0 3.64 ± 0.11 23.3 ± 7.94 ± 0.25 0.37 ± 0.17 ± 35.4 ± 8.38 ± 21.5 ± 0.28 3.87 ±
0.32bcde 0.01abc 0.01abc 0.37abcde 0.13 0.10abc
12 1 3.50 ± 0.09 23.7 ± 7.54 ± 0.32 0.34 ± 0.14 ± 35.2 ± 7.82 ± 20.9 ± 0.43 3.07 ±
0.50bcd 0.02abc 0.01abc 0.53abcde 0.08 0.22def
12 4 3.49 ± 0.11 21.7 ± 7.73 ± 0.27 0.35 ± 0.16 ± 33.5 ± 8.09 ± 21.0 ± 0.50 3.90 ±
0.42de 0.02abc 0.02abc 0.66cdef 0.11 0.15abc
12 9 3.62 ± 0.08 22.4 ± 7.23 ± 0.04 0.36 ± 0.17 ± 33.8 ± 8.73 ± 20.1 ± 0.50 3.76 ±
0.71cde 0.02abc 0.02abc 0.82bcdef 0.09 0.13abcd
12 16 3.68 ± 0.18 22.2 ± 7.19 ± 0.26 0.36 ± 0.16 ± 33.6 ± 8.73 ± 20.1 ± 0.37 4.02 ±
0.30cde 0.01abc 0.02abc 0.66bcdef 0.13 0.08ab
12 31 3.69 ± 0.13 26.3 ± 7.51 ± 0.43 0.37 ± 0.18 ± 0.00ab 38.0 ± 1.50a 8.85 ± 19.2 ± 0.74 3.12 ±
0.96a 0.01abc 0.01 0.27cdef
16 0 4.05 ± 0.08 22.4 ± 7.26 ± 0.25 0.33 ± 0.01c 0.12 ± 0.01 c
34.2 ± 8.63 ± 21.7 ± 0.23 4.09 ± 0.08a
0.17cde 0.23bcdef 0.25
16 1 3.80 ± 0.19 21.0 ± 6.60 ± 0.11 0.32 ± 0.12 ± 0.01c 31.8 ± 0.47f 8.68 ± 20.3 ± 0.38 4.15 ± 0.13a
0.29e 0.01abc 0.13
16 4 3.83 ± 0.07 21.7 ± 6.77 ± 0.16 0.34 ± 0.16 ± 32.8 ± 0.65ef 8.74 ± 20.8 ± 0.56 4.22 ± 0.09a
0.51de 0.01abc 0.00abc 0.30
16 9 3.98 ± 0.14 22.2 ± 6.46 ± 0.27 0.33 ± 0.13 ± 0.02bc 33.1 ± 8.84 ± 20.1 ± 0.23 4.05 ±
0.34cde 0.01abc 0.65def 0.30 0.08ab
16 16 4.06 ± 0.13 21.8 ± 6.65 ± 0.16 0.31 ± 0.01bc 0.15 ± 33.1 ± 9.04 ± 19.7 ± 0.25 4.04 ±
0.55de 0.01abc 0.85def 0.17 0.09ab
16 31 3.70 ± 0.13 25.4 ± 6.16 ± 0.16 0.38 ± 0.01ab 0.20 ± 0.00a 35.8 ± 9.03 ± 18.9 ± 0.13 3.60 ±
0.18ab 0.37abcde 0.30 0.04abcde
Pooled SE 0.038 0.167 0.115 0.004 0.003 0.223 0.060 0.170 0.066
Means of main effect
Lipid level Starvation
(%) day
0 3.67 ± 0.09 23.2 ± 8.25 ± 0.26 0.36 ± 0.01A 0.15 ± 35.67 ± 8.34 ± 21.9 ± 3.73 ± 0.11A
0.22B A
0.01AB 0.36AB 0.12BC 0.27A
1 3.49 ± 0.09 23.1 ± 7.71 ± 0.32 ± 0.01C 0.14 ± 0.01B 34.7 ± 8.09 ± 21.6 ± 3.15 ± 0.23C
0.46B 0.28AB 0.63BC 0.14C 0.43ABC
4 3.56 ± 0.07 22.5 ± 7.78 ± 0.34 ± 0.16 ± 34.3 ± 8.35 ± 21.8 ± 3.65 ±
0.37B 0.26AB 0.01ABC 0.00AB 0.56BC 0.13BC 0.44AB 0.18AB
9 3.66 ± 0.11 22.7 ± 7.39 ± 0.34 ± 0.15 ± 34.3 ± 8.67 ± 20.8 ± 3.70 ±
0.32B 0.29BC 0.01ABC 0.01AB 0.53BC 0.13AB 0.36BC 0.12AB
16 3.67 ± 0.11 22.5 ± 7.34 ± 0.33 ± 0.16 ± 34.1 ± 0.43C 8.78 ± 20.7 ± 3.65 ±
0.29B 0.21BC 0.01BC 0.01AB 0.12AB 0.38BC 0.15AB
31 3.59 ± 0.08 25.2 ± 0.47 6.97 ± 0.28 0.35 ± 0.17 ± 0.01A 36.3 ± 0.66A 8.95 ± 20.1 ± 3.34 ±
A C
0.01AB 0.13A 0.46C 0.15BC
8 3.31 ± 24.0 ± 8.57 ± 0.34 ± 0.01Y 0.16 ± 0.00 36.3 ± 0.26X 8.31 ± 22.6 ± 2.99 ± 0.09Z
0.04Z 0.18X 0.16X 0.10Y 0.23X
12 3.60 ± 23.2 ± 7.55 ± 0.36 ± 0.01X 0.16 ± 0.01 34.8 ± 0.39Y 8.41 ± 20.6 ± 3.67 ± 0.09Y
0.05Y 0.33Y 0.11Y 0.08Y 0.22Y
16 3.92 ± 22.4 ± 6.69 ± 0.34 ± 0.01Y 0.15 ± 0.01 33.5 ± 0.31Z 8.82 ± 20.4 ± 4.04 ± 0.05X
0.05X 0.28Z 0.10Z 0.10X 0.21Y
P-values of two-way ANOVA
Starvation 0.469 0.000 * 0.000 * 0.002 * 0.015 * 0.000 * 0.000 * 0.000 * 0.000 *
Lipid level 0.000 * 0.000 * 0.000 * 0.004 * 0.054 0.000 * 0.000 * 0.000 * 0.000 *
Starvation × Lipid level 0.621 0.000 * 0.062 0.034 * 0.006 * 0.001 * 0.729 0.607 0.001 *

Variable C22:1n-9 C24:1n-9 MUFA C18:2n-6 C18:3n-6 C20:2n-6 C20:3n-6 C20:4n-6 C22:2n-6
Table 5 (-continued) Two-way ANOVA for effects of starvation and dietary lipid level on liver fatty acid composition of experimental tiger puffer (% total fatty acids, mean ± standard
error)
Individual treatment
(continued on next page)

8
Z. Liao et al. Aquaculture Reports 24 (2022) 101095

Table 5 (continued )
Variable C14:0 C16:0 C18:0 C20:0 C22:0 SFA C16:1n-7 C18:1n-9 C20:1n-9

Lipid level Starvation

(%) day
8 0 2.03 ± 0.54 ± 0.03 36.4 ± 10.4 ± 0.30 0.11 ± 0.68 ± 0.06 0.05 ± 0.28 ± 0.21 ± 0.01a
0.08def 0.48abc 0.01bcdefg 0.00c 0.01c
8 1 1.68 ± 0.11f 0.44 ± 0.03 35.5 ± 10.6 ± 0.21 0.10 ± 0.58 ± 0.03 0.05 ± 0.26 ± 0.17 ±
0.53bcd 0.00efgh 0.00b 0.01c 0.01ab
8 4 1.81 ± 0.46 ± 0.01 36.9 ± 9.91 ± 0.23 0.07 ± 0.00h 0.62 ± 0.02 0.05 ± 0.27 ± 0.18 ±
0.06ef 0.49ab 0.00b 0.01c 0.00ab
8 9 1.80 ± 0.54 ± 0.03 36.4 ± 10.2 ± 0.14 0.10 ± 0.72 ± 0.05 0.06 ± 0.28 ± 0.22 ± 0.01a
0.10ef 0.32abc 0.00cdefg 0.01b 0.02c
8 16 1.78 ± 0.49 ± 0.03 36.0 ± 10.3 ± 0.26 0.10 ± 0.65 ± 0.04 0.06 ± 0.28 ± 0.19 ±
0.11ef 0.51abcd 0.00defg 0.00b 0.01bc 0.01ab
8 31 2.29 ± 0.56 ± 0.05 36.6 ± 9.52 ± 0.06 0.11 ± 0.63 ± 0.05 0.06 ± 0.32 ± 0.20 ±
0.26cdef 0.51abc 0.01cdefg 0.01b 0.04abc 0.02ab
12 0 2.70 ± 0.61 ± 0.03 37.1 ± 10.1 ± 0.17 0.12 ± 0.58 ± 0.02 0.05 ± 0.33 ± 0.19 ±
0.10abc 0.19ab 0.00abcde 0.00c 0.01abc 0.01ab
12 1 2.24 ± 0.45 ± 0.04 34.5 ± 10.2 ± 0.49 0.12 ± 0.57 ± 0.05 0.07 ± 0.35 ± 0.15 ± 0.01b
0.16cdef 0.61cd 0.00abcdef 0.00b 0.01abc
12 4 2.70 ± 0.63 ± 0.07 36.3 ± 9.87 ± 0.28 0.08 ± 0.00gh 0.60 ± 0.03 0.07 ± 0.38 ± 0.19 ±
0.07abc 0.53abcd 0.00b 0.01ab 0.01ab
12 9 2.56 ± 0.63 ± 0.04 35.7 ± 10.0 ± 0.15 0.12 ± 0.66 ± 0.05 0.06 ± 0.36 ± 0.20 ±
0.12abcd 0.41abcd 0.00abcde 0.00b 0.01abc 0.02ab
12 16 2.65 ± 0.60 ± 0.04 36.1 ± 9.81 ± 0.09 0.13 ± 0.68 ± 0.02 0.07 ± 0.31 ± 0.20 ±
0.07abcd 0.48abcd 0.01abcd 0.00b 0.02abc 0.01ab
12 31 2.34 ± 0.65 ± 0.11 34.1 ± 9.76 ± 0.35 0.12 ± 0.71 ± 0.04 0.07 ± 0.28 ± 0.20 ±
0.34bcde 0.06d 0.01abcde 0.01ab 0.04c 0.00ab
16 0 2.97 ± 0.56 ± 0.03 37.9 ± 9.36 ± 0.24 0.10 ± 0.54 ± 0.02 0.07 ± 0.40 ± 0.17 ±
0.04ab 0.24a 0.01efgh 0.00ab 0.01a 0.00ab
16 1 3.06 ± 0.58 ± 0.05 36.8 ± 9.79 ± 0.12 0.14 ± 0.01ab 0.64 ± 0.05 0.09 ± 0.37 ± 0.19 ±
0.13a 0.30ab 0.02a 0.02ab 0.02ab
16 4 3.12 ± 0.63 ± 0.05 37.5 ± 9.25 ± 0.26 0.09 ± 0.57 ± 0.03 0.07 ± 0.40 ± 0.19 ±
0.10a 0.35ab 0.00fgh 0.00ab 0.01a 0.01ab
16 9 2.87 ± 0.56 ± 0.02 36.4 ± 9.26 ± 0.18 0.13 ± 0.61 ± 0.01 0.07 ± 0.38 ± 0.18 ±
0.08abc 0.44abc 0.00abc 0.00ab 0.01ab 0.00ab
16 16 2.96 ± 0.62 ± 0.03 36.6 ± 9.25 ± 0.16 0.13 ± 0.64 ± 0.03 0.07 ± 0.39 ± 0.19 ±
0.08ab 0.39abc 0.01abc 0.00ab 0.02a 0.01ab
16 31 3.19 ± 0.80 ± 0.02 35.5 ± 9.09 ± 0.21 0.14 ± 0.00a 0.68 ± 0.02 0.07 ± 0.31 ± 0.21 ± 0.01a
0.09a 0.14bcd 0.00ab 0.03abc
Pooled SE 0.061 0.012 0.132 0.070 0.003 0.009 0.003 0.066 0.003
Means of main effect
Lipid level Starvation
(%) day
0 2.56 ± 0.11 0.57 ± 37.2 ± 0.23 9.95 ± 0.11 ± 0.60 ± 0.02B 0.03 ± 0.34 ± 0.01 0.19 ±
0.02B A
0.17AB 0.00AB 0.01B 0.01AB
1 2.28 ± 0.18 0.49 ± 35.6 ± 10.2 ± 0.18A 0.11 ± 0.01B 0.60 ± 0.02B 0.07 ± 0.32 ± 0.02 0.17 ± 0.01B
0.03B 0.37B 0.01A
C B
4 2.53 ± 0.16 0.57 ± 36.9 ± 9.66 ± 0.08 ± 0.00 0.60 ± 0.02 0.06 ± 0.35 ± 0.02 0.19 ±
0.03B 0.28A 0.16AB 0.00A 0.00AB
A
9 2.45 ± 0.14 0.58 ± 36.2 ± 9.80 ± 0.12 ± 0.66 ± 0.02 0.06 ± 0.34 ± 0.01 0.20 ±
0.02AB 0.23AB 0.15AB 0.00AB 0.00A 0.01AB
16 2.45 ± 0.15 0.57 ± 36.2 ± 9.80 ± 0.12 ± 0.66 ± 0.02A 0.07 ± 0.33 ± 0.02 0.19 ±
0.02B 0.26AB 0.16AB 0.01AB 0.00A 0.00AB
31 2.60 ± 0.18 0.67 ± 0.04 35.6 ± 9.43 ± 0.13B 0.12 ± 0.01A 0.67 ± 0.02A 0.07 ± 0.31 ± 0.02 0.20 ± 0.01A
A
0.36B 0.00A
X Y
8 1.90 ± 0.51 ± 36.3 ± 10.2 ± 0.11 0.10 ± 0.00 0.65 ± 0.02 0.05 ± 0.28 ± 0.19 ± 0.01
0.06Z 0.01Y 0.20Y 0.00Y 0.01Z
12 2.55 ± 0.59 ± 35.8 ± 9.99 ± 0.10X 0.12 ± 0.00X 0.62 ± 0.02 0.05 ± 0.34 ± 0.19 ± 0.01
0.06Y 0.02X 0.26Y 0.01Y 0.01Y
16 3.02 ± 0.62 ± 36.9 ± 9.33 ± 0.09Y 0.12 ± 0.00X 0.61 ± 0.01 0.08 ± 0.38 ± 0.18 ± 0.00
0.04X 0.02X 0.19X 0.00X 0.01X
P-values of two-way ANOVA
Starvation 0.087 0.000 * 0.000 * 0.015 * 0.000 * 0.015 * 0.000 * 0.103 0.022 *
Lipid level 0.000 * 0.000 * 0.000 * 0.000 * 0.000 * 0.332 0.000 * 0.000 * 0.323
Starvation × Lipid level 0.047 * 0.051 0.026 * 0.933 0.001 * 0.086 0.000 * 0.002 * 0.028 *

Variable n-6PUFA C18:3n-3 C20:3n-3 C20:5n-3 C22:5n-3 C22:6n-3 n-3PUFA n-3/n-6

Table 5 (-continued) Two-way ANOVA for effects of starvation and dietary lipid level on liver fatty acid composition of experimental tiger puffer (% total fatty acids, mean ± standard
error)
Individual treatment
Lipid level Starvation
(%) day
8 0 11.7 ± 0.37 1.58 ± 0.04 0.27 ± 0.02 3.04 ± 3.49 ± 0.12 7.93 ± 0.15 16.3 ± 1.40 ± 0.04
0.09defg 0.19
8 1 11.7 ± 0.23 1.49 ± 0.06 0.26 ± 0.01 3.10 ± 3.33 ± 0.11 7.86 ± 0.27 16.0 ± 1.37 ± 0.03
0.12cdefg 0.36
(continued on next page)

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Z. Liao et al. Aquaculture Reports 24 (2022) 101095

Table 5 (continued )
Variable C14:0 C16:0 C18:0 C20:0 C22:0 SFA C16:1n-7 C18:1n-9 C20:1n-9

8 4 11.1 ± 0.24 1.55 ± 0.01 0.24 ± 0.01 2.92 ± 3.21 ± 0.10 7.56 ± 0.24 15.5 ± 1.40 ± 0.05
0.09efgh 0.42
8 9 11.6 ± 0.21 1.55 ± 0.02 0.26 ± 0.02 2.73 ± 0.05gh 3.52 ± 0.17 7.73 ± 0.28 15.8 ± 1.36 ± 0.04
0.40
8 16 11.6 ± 0.29 1.57 ± 0.03 0.26 ± 0.01 2.95 ± 3.62 ± 0.09 8.61 ± 0.33 17.0 ± 1.47 ± 0.05
0.03efgh 0.43
8 31 10.8 ± 0.04 1.52 ± 0.06 0.27 ± 0.01 3.06 ± 3.35 ± 0.07 8.84 ± 0.78 17.0 ± 1.57 ± 0.12
0.41defg 1.28
12 0 11.4 ± 0.17 1.60 ± 0.13 0.27 ± 0.01 3.71 ± 3.39 ± 0.04 9.64 ± 0.19 18.6 ± 1.64 ± 0.04
0.08abcde 0.27
12 1 11.5 ± 0.55 1.65 ± 0.02 0.29 ± 0.02 3.58 ± 3.92 ± 0.44 9.32 ± 0.29 18.8 ± 1.64 ± 0.05
0.10abcdef 0.37
12 4 11.2 ± 0.33 1.61 ± 0.04 0.26 ± 0.02 3.86 ± 3.48 ± 0.11 9.84 ± 0.35 19.0 ± 1.70 ± 0.02
0.10abcd 0.52
12 9 11.4 ± 0.15 1.69 ± 0.05 0.28 ± 0.01 3.45 ± 3.66 ± 0.06 9.95 ± 0.39 19.0 ± 1.67 ± 0.06
0.30bcdefg 0.65
12 16 11.2 ± 0.09 1.64 ± 0.02 0.29 ± 0.01 3.06 ± 3.99 ± 0.21 10.2 ± 0.36 19.2 ± 1.71 ± 0.04
0.12defg 0.45
12 31 11.1 ± 0.28 1.32 ± 0.08 0.26 ± 0.04 2.19 ± 0.14h 4.09 ± 0.46 8.85 ± 1.54 16.7 ± 1.51 ± 0.20
1.82
16 0 10.6 ± 0.25 1.63 ± 0.11 0.25 ± 0.00 4.31 ± 0.07a 3.35 ± 0.03 10.6 ± 0.14 20.1 ± 1.90 ± 0.06
0.14
16 1 11.2 ± 0.09 1.79 ± 0.02 0.28 ± 0.01 4.10 ± 0.12ab 3.66 ± 0.21 10.3 ± 0.30 20.2 ± 1.80 ± 0.07
0.63
ab
16 4 10.6 ± 0.25 1.66 ± 0.05 0.24 ± 0.04 4.06 ± 0.16 3.27 ± 0.11 9.86 ± 0.25 19.1 ± 1.81 ± 0.06
0.37
16 9 10.6 ± 0.18 1.71 ± 0.03 0.26 ± 0.00 3.92 ± 3.47 ± 0.09 10.5 ± 0.23 19.8 ± 1.87 ± 0.06
0.11abc 0.40
16 16 10.5 ± 0.05 1.69 ± 0.04 0.27 ± 0.01 3.82 ± 3.65 ± 0.06 10.5 ± 0.45 19.9 ± 1.89 ± 0.07
0.10abcd 0.73
16 31 10.5 ± 0.21 1.50 ± 0.03 0.32 ± 0.01 2.85 ± 3.40 ± 0.04 10.1 ± 0.17 18.1 ± 1.73 ± 0.04
0.14fgh 0.15
Pooled SE 0.071 0.017 0.004 0.068 0.040 0.140 0.210 0.024
Means of main effect
Lipid level Starvation
(%) day
0 11.2 ± 0.18 1.60 ± 0.26 ± 0.01 3.69 ± 0.13A 3.41 ± 9.37 ± 0.28 18.3 ± 1.65 ± 0.05
0.06AB 0.04AB 0.39
1 11.5 ± 0.18 1.63 ± 0.27 ± 0.01 3.56 ± 3.61 ± 9.07 ± 0.33 18.2 ± 1.58 ± 0.06
0.04A 0.13AB 0.16AB 0.56
4 10.9 ± 0.16 1.61 ± 0.24 ± 0.01 3.59 ± 3.31 ± 0.06B 9.03 ± 0.34 17.8 ± 1.63 ± 0.06
0.02AB 0.16AB 0.53
9 11.2 ± 0.16 1.66 ± 0.03 0.27 ± 0.01 3.41 ± 3.55 ± 9.47 ± 0.38 18.4 ± 1.65 ± 0.07
A
0.17AB 0.06AB 0.56
16 11.2 ± 0.17 1.63 ± 0.02 0.27 ± 0.01 3.29 ± 0.12B 3.73 ± 0.08A 9.74 ± 0.31 18.5 ± 1.67 ± 0.06
A
0.46
C
31 10.8 ± 0.12 1.46 ± 0.28 ± 0.01 2.77 ± 0.20 3.55 ± 9.25 ± 0.48 17.3 ± 1.61 ± 0.07
0.04B 0.14AB 0.66
Z
8 11.4 ± 1.55 ± 0.26 ± 2.98 ± 0.07 3.42 ± 0.05Y 8.10 ± 0.17Z
16.3 ± 1.43 ±
0.12X 0.02Y 0.00Y 0.25Z 0.03Z
12 11.3 ± 1.60 ± 0.27 ± 3.38 ± 0.12Y 3.71 ± 0.10X 9.66 ± 0.20Y 18.6 ± 1.65 ±
0.11X 0.04XY 0.01X 0.28Y 0.03Y
16 10.67 ± 1.66 ± 0.27 ± 3.89 ± 0.09X 3.46 ± 0.05Y 10.3 ± 0.11X 19.6 ± 1.84 ±
0.09Y 0.03X 0.01X 0.20X 0.03X
P-values of two-way ANOVA
Starvation 0.064 0.009 * 0.087 0.000 * 0.009 * 0.375 0.110 0.652
Lipid level 0.000 * 0.010 * 0.023 * 0.000 * 0.001 * 0.000 * 0.000 * 0.000 *
Starvation × Lipid level 0.861 0.352 0.370 0.000 * 0.314 0.347 0.097 0.182

Data in a same column not sharing a same superscript letter were significantly different (P < 0.05). * : P < 0.05. SFA: saturated fatty acid.
Data in a same column not sharing a same superscript letter were significantly different (P < 0.05). * : P < 0.05. MUFA: monounsaturated fatty acid.
Data in a same column not sharing a same superscript letter were significantly different (P < 0.05). * : P < 0.05. PUFA: Polyunsaturated fatty acid.

unique structural and physiological roles in polar lipids, are less Its dietary content is usually low, and thus the majority of DPA in fish
responsive to dietary changes and can be selectively retained during tissues is derived from the elongation of EPA, or possibly also from
starvation (Sargent et al., 2002). There were two other interesting retro-conversion of DHA (Tocher, 2003; Xu et al., 2020). The elevation
findings about the response of muscle fatty acid composition to starva­ of muscle DPA content by starvation could be related to the attempt of
tion. The first was that the contents of two n-6 fatty acids, C18:3n-6 and fish to biosynthesize DHA to compensate the DHA loss during starvation.
ARA, were increased by starvation. A previous study with tiger puffer A recent meta-analysis study indicates an apparent appearance of DPA
showed that n-6 fatty acids could not be efficiently utilized in this fish in marine fish, likely, owing to the elongation of EPA. However,
(Xu et al., 2019; Yu et al., 2019). This may explain why the n-6 fatty considering the majority of marine fish lack the capacity to efficiently
acids were retained during starvation. Another interesting finding was express Elovl2 and Δ6 desaturase enzymes, further bioconversion of
that the muscle DPA content was drastically increased by starvation. DPA to DHA could be limited (Tocher et al., 2006; Geay et al., 2010;
DPA is known to be an intermediate of the DHA biosynthetic pathway. Castro et al., 2016). In addition, the increase of DPA by starvation was

10
Z. Liao et al. Aquaculture Reports 24 (2022) 101095

Table 6 more significant when fish were previously fed higher-lipid diets. This
Two-way ANOVA for effects of starvation and dietary lipid level on tissues could be due to more adequate supply of EPA substrate.
biochemical parameters of experimental tiger puffer (mean ± standard error). Similar to what observed in muscle, starvation also did not exert
Parameter Muscle Liver great influence on the liver fatty acid composition. Nevertheless,
Glycogen Glycogen TC MDA
C18:1n-9 seemed to be primarily mobilized for energy purpose during
(mg/g) (mg/g) (mmol/g) (μmol/g) starvation. Alongside with the high C16:1n-7 (approximately 8%) and
C18:1n-9 (approximately 22%) contents in the liver, this result indicates
Individual treatment
Lipid Starvation that the liver of tiger puffer has typical characteristics of lipid-storage
level day tissue. The liver C18:0 content was also decreased by starvation.
(%) C18:0 is present in relatively small quantities in fish tissues and is
8 0 2.22 ± 0.34 47.6 ± 5.27 2.17 ± 14.7 ± generally considered a poor substrate for triacylglycerol synthesis and
0.07c 0.82f
8 1 2.26 ± 0.90 25.8 ± 4.13 2.44 ± 14.7 ±
energy mobilization (Henderson, 1996; Johnsen et al., 2000). In this
0.13abc 0.14f study, parts of the C18:0 in the liver could be converted into C18:1n-9
8 4 0.91 ± 0.26 10.3 ± 1.13 2.87 ± 18.1 ± for energy supply. Moreover, a recent study with tiger puffer showed
0.13abc 0.79ef that this fish species have a high C18:0 content in the muscle (Xu et al.,
8 9 1.38 ± 0.26 9.70 ± 1.39 2.47 ± 20.7 ±
2021). The liver C18:0 could be transferred to muscle for special func­
0.29abc 0.84de
8 16 0.50 ± 0.16 9.14 ± 0.71 2.09 ± 23.6 ± tion demands during starvation. Starvation resulted in only a slight
0.06c 2.75cde decrease of LC-PUFA, mostly EPA, in the liver, suggesting that the liver
8 31 0.55 ± 0.04 8.85 ± 1.04 2.08 ± 18.8 ± of tiger puffer has a high buffering capacity in response to feed depri­
0.11c 0.80def vation. EPA seemed to be less important than DHA in the maintenance of
12 0 1.87 ± 0.58 44.1 ± 5.65 2.38 ± 21.6 ±
0.14bc 0.59cde
membrane structure. In Atlantic salmon, when deprived of dietary n-3
12 1 2.41 ± 0.45 37.5 ± 4.37 2.41 ± 20.1 ± LC-PUFA, phospholipids in fish show selective retention of DHA, despite
0.14abc 1.68def being completely devoid of EPA (Thomassen et al., 2017), and when in
12 4 0.94 ± 0.17 20.5 ± 1.80 2.35 ± 23.6 ± dietary surplus, EPA can be extensively β-oxidised (Codabaccus et al.,
0.05bc 0.96cd
2012).
12 9 0.92 ± 0.21 10.0 ± 0.29 2.57 ± 23.5 ±
0.31abc 1.08cde The change of other nutrients in muscle and liver can well explained
12 16 0.60 ± 0.32 10.1 ± 0.24 2.83 ± 30.2 ± the lipid-related composition, although these parameters are not
0.15abc 0.66ab commonly concerned from the perspective of food function. Glycogen is
12 31 0.48 ± 0.08 10.2 ± 1.41 2.57 ± 20.4 ± an essential fuel mainly stored in the liver, and when required, is
0.13abc 0.71de
16 0 2.52 ± 0.55 38.0 ± 3.75 2.31 ± 23.3 ±
enzymatically broken down and transported to the extrahepatic tissues
0.08c 0.22cde as glucose. Liver glycogen appear to be the primary and early source of
16 1 2.65 ± 0.37 28.9 ± 3.35 2.58 ± 23.4 ± energy during fasting (Navarro and Gutiérrez, 1995). This was consis­
0.09abc 1.09cde tent with results of the present study, which showed that starvation for 4
16 4 1.51 ± 0.24 11.6 ± 0.99 3.06 ± 26.7 ±
days dramatically decreased the liver glycogen stores and later at slower
0.37abc 0.10bc
16 9 1.01 ± 0.21 9.12 ± 0.44 3.48 ± 30.3 ± rates. This happened especially when fish were fed control diets and thus
0.45a 1.25ab had a lower energy storage. Similar results were found in channel catfish
16 16 1.16 ± 0.24 9.40 ± 0.65 3.40 ± 34.6 ± (Gaylord and Gatlin, 2000), sunshine bass (Morone chrysops × Morone
0.39ab 0.26a saxatilis) (Davis and Gaylord, 2011) and zebrafish (Lu et al., 2019). The
16 31 0.29 ± 0.03 10.5 ± 0.21 2.38 ± 20.9 ±
concentration of glycogen is very low in the muscle, and its contribution
0.11bc 0.23de
Pooled SE (One-way 0.114 1.667 0.062 0.721 to energy expenditure of starved animals is also relatively limited (Love,
ANOVA) 1970). The muscle glycogen mobilization is likely related to an increase
Means of main effect in muscular activity than to the fasting process (Navarro and Gutiérrez,
Lipid Starvation
1995). In the current study, muscle glycogen content was quite low and
level day
(%) decreased after starved for 1 day, which was similar to what observed in
0 2.20 ± 43.2 ± 2.28 ± 19.9 ± other fish species such as Pacific bluefin tuna (Miyake et al., 2011). In
0.28A 2.86A 0.06B 1.35C addition, although not as energy substance, the concentration of
1 2.44 ± 30.2 ± 2.48 ± 19.4 ± cholesterol and malondialdehyde, product of lipid peroxidation, in the
0.35A 2.47B 0.07AB 1.39C
liver of tiger puffer were positively correlated with the liver lipid con­
4 1.13 ± 14.1 ± 2.76 ± 22.8 ±
0.15B 1.41C 0.15AB 1.31B tent, suggesting that cautions should be paid on other lipid-related
9 1.11 ± 9.58 ± 2.84 ± 24.8 ± biological parameters if liver is consumed (Regost et al., 2001; Du
0.13B 0.50C 0.23A 1.53B et al., 2005).
16 0.76 ± 9.51 ± 2.77 ± 29.4 ±
Muscle texture was the final quality trait exhibiting to the con­
0.15B 0.34C 0.19AB 1.80A
31 0.44 ± 9.85 ± 2.34 ± 20.1 ±
sumers, and it can be influenced by mixed factors. Unexpectedly, star­
0.04B 0.62C 0.08AB 0.44C vation for 1 day resulted in that all the texture parameters reached the
8 1.31 ± 0.20 20.2 ± 3.13 2.35 ± 18.4 ± maximum value. Increased hardness by short-term starvation was also
0.08Y 0.88Z observed in gilthead sea bream (Ginés et al., 2002), but this was quite
12 1.20 ± 0.19 23.6 ± 3.12 2.52 ± 23.2 ±
difficult to explain. The hardness was assumed to be primarily related to
0.07Y 0.89Y
16 1.52 ± 0.20 19.1 ± 2.42 2.87 ± 26.5 ± the lipid content (Ruiz-Carrascal et al., 2000; Rasmussen, 2001; John­
0.14X 1.16X ston et al., 2006), but this was not observed in the present study.
P-values of two-way ANOVA Additional swimming exercise of tiger puffer during the early starved
Starvation 0.000 * 0.000 * 0.004 * 0.000 *
days may contribute to the accelerated lipid catabolism and increase of
Lipid level 0.326 0.068 0.000 * 0.000 *
Starvation × Lipid level 0.914 0.250 0.017 * 0.008 *
hardness. Nevertheless, increase of muscle hardness by long-term star­
vation has also been observed in Atlantic salmon (Einen and Thomassen,
Data in a same column not sharing a same superscript letter were significantly 1998) and Atlantic cod (Gadus morhua) (Bjørnevik et al., 2017). This was
different (P < 0.05). * : P < 0.05.
different from the present study, which showed that long-term starva­
tion resulted in decrease in not only hardness, but also other texture

11
Z. Liao et al. Aquaculture Reports 24 (2022) 101095

Table 7
Two-way ANOVA for effects of starvation and dietary lipid level on muscle texture of experimental tiger puffer (mean ± standard error).
Variable Hardness (N) Adhesiveness (mJ) Cohesiveness (Ratio) Springiness (mm) Gumminess (N) Chewiness (mJ)

Individual treatment
Lipid level (%) Starvation day
8 0 2.43 ± 0.11bcd 0.028 ± 0.003 0.41 ± 0.02 1.26 ± 0.10 1.03 ± 0.07bcd 1.43 ± 0.18
8 1 3.23 ± 0.28ab 0.028 ± 0.002 0.40 ± 0.01 1.54 ± 0.09 1.33 ± 0.14abc 2.21 ± 0.36
8 4 2.98 ± 0.16abc 0.026 ± 0.002 0.41 ± 0.01 1.42 ± 0.07 1.23 ± 0.09abc 1.79 ± 0.23
8 9 2.65 ± 0.09bcd 0.028 ± 0.002 0.39 ± 0.02 1.36 ± 0.11 1.03 ± 0.08bcd 1.45 ± 0.23
8 16 2.84 ± 0.19abc 0.028 ± 0.002 0.39 ± 0.01 1.25 ± 0.03 1.09 ± 0.09bcd 1.41 ± 0.15
12 0 2.20 ± 0.21cd 0.023 ± 0.002 0.43 ± 0.02 1.11 ± 0.10 0.94 ± 0.11bcd 1.15 ± 0.21
12 1 3.69 ± 0.18a 0.025 ± 0.003 0.42 ± 0.01 1.49 ± 0.08 1.59 ± 0.11a 2.57 ± 0.34
12 4 3.08 ± 0.26abc 0.020 ± 0.000 0.38 ± 0.01 1.31 ± 0.04 1.20 ± 0.10abc 1.66 ± 0.15
12 9 2.22 ± 0.18cd 0.025 ± 0.003 0.39 ± 0.03 1.20 ± 0.08 0.88 ± 0.12cd 1.13 ± 0.27
12 16 1.86 ± 0.25d 0.025 ± 0.005 0.37 ± 0.02 0.95 ± 0.10 0.68 ± 0.07d 0.70 ± 0.13
16 0 2.93 ± 0.21abc 0.026 ± 0.002 0.42 ± 0.01 1.40 ± 0.09 1.23 ± 0.10abc 1.79 ± 0.23
16 1 2.86 ± 0.18abc 0.030 ± 0.000 0.43 ± 0.01 1.64 ± 0.15 1.25 ± 0.09abc 2.11 ± 0.28
16 4 3.37 ± 0.17ab 0.030 ± 0.000 0.41 ± 0.01 1.46 ± 0.04 1.40 ± 0.07ab 2.17 ± 0.15
16 9 2.78 ± 0.16abcd 0.028 ± 0.004 0.39 ± 0.01 1.23 ± 0.06 1.12 ± 0.07abcd 1.51 ± 0.18
16 16 2.51 ± 0.16bcd 0.028 ± 0.002 0.34 ± 0.02 1.03 ± 0.10 0.89 ± 0.08cd 1.03 ± 0.20
Pooled SE (One-way ANOVA) 0.068 0.001 0.005 0.030 0.033 0.077
Means of main effect
Lipid level (%) Starvation day
0 2.52 ± 0.12B 0.026 ± 0.001 0.42 ± 0.01A 1.26 ± 0.06BC 1.06 ± 0.06BC 1.46 ± 0.13BC
1 3.23 ± 0.15A 0.028 ± 0.001 0.42 ± 0.01A 1.56 ± 0.06A 1.38 ± 0.07A 2.27 ± 0.18A
4 3.15 ± 0.11A 0.026 ± 0.001 0.40 ± 0.01AB 1.40 ± 0.03AB 1.28 ± 0.05AB 1.89 ± 0.12AB
9 2.58 ± 0.10B 0.027 ± 0.002 0.39 ± 0.01AB 1.27 ± 0.05BC 1.02 ± 0.05C 1.38 ± 0.13BC
16 2.45 ± 0.15B 0.027 ± 0.002 0.37 ± 0.01B 1.09 ± 0.06C 0.90 ± 0.06C 1.07 ± 0.12C
8 2.81 ± 0.09XY 0.028 ± 0.001X 0.40 ± 0.01 1.36 ± 0.04X 1.14 ± 0.05 1.65 ± 0.12
12 2.57 ± 0.17Y 0.024 ± 0.001Y 0.40 ± 0.01 1.20 ± 0.05Y 1.05 ± 0.08 1.41 ± 0.16
16 2.89 ± 0.09X 0.028 ± 0.001X 0.40 ± 0.01 1.35 ± 0.06X 1.18 ± 0.05 1.72 ± 0.12
P-values of two-way ANOVA
Starvation 0.000 * 0.779 0.001 * 0.000 * 0.000 * 0.000 *
Lipid level 0.070 0.011 * 0.928 0.020 * 0.163 0.159
Starvation × Lipid level 0.001 * 0.866 0.437 0.513 0.015 * 0.251

Data in a same column not sharing a same superscript letter were significantly different (P < 0.05). * : P < 0.05.

parameters such as cohesiveness, springiness and chewiness. These re­ Declaration of Competing Interest
sults suggested the complexity of relationship between muscle texture
and starvation. Various relationships between chemical composition The authors declare that they have no known competing financial
attributes and springiness, cohesiveness or chewiness have been re­ interests or personal relationships that could have appeared to influence
ported in aquaculture species such as catfish (Bland et al., 2018), tilapia the work reported in this paper.
(Oreochromis niloticus) (Santos et al., 2021), sea bream (Orban et al.,
1997), and blackspot seabream (Pagellus bogaraveo) (Rincón et al., Data availability statement
2016). Nevertheless, it was indicative from this study that excessive
starvation caused adverse effects on fillet texture of tiger puffer, whereas Raw data supporting the conclusions of this manuscript will be made
shorter-term (1–4 days) may have beneficial effects, irrespective of lipid available by the authors, without undue reservation, to any qualified
accumulation in fish body. Customed fillet texture may be obtained by researcher.
manipulation of starvation time.
Acknowledgments
5. Conclusion
This work was supported by National Key R&D Program of China
In farmed tiger puffer, the muscle lipid content decreased linearly (2018YFD0900400), China Agriculture Research System of MOF and
with starvation time, but the liver lipid content firstly increased (from 1 MARA (CARS-47), and Central Public-interest Scientific Institution Basal
to 16 days of starvation), and thereafter sharply declined. Starvation Research Fund, YSFRI/CAFS (2020TD48, 20603022022004).
primarily mobilized C14:0, C16:1n-7, C20:1n-9 and C22:1n-9 in the
muscle, and C18:1n-9 and C20:5n-3 in the liver, but it largely increased Appendix A. Supporting information
the C22:5n-3 content in the muscle. Interactive effects between starva­
tion time and dietary lipid level were observed for many fatty acids. Supplementary data associated with this article can be found in the
Long-term (31 days) starvation could cause adverse effects on fillet online version at doi:10.1016/j.aqrep.2022.101095.
texture of tiger puffer, whereas shorter-term starvation (1–4 days) may
have beneficial effects, irrespective of lipid accumulation in fish body. References

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