AACL Bioflux, 2021, Volume 14, Issue 2.
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Water temperature and stocking density for long-
hour transportation of hybrid grouper
Epinephelus fuscoguttatus x E. lanceolatus
Norfazreena M. Faudzi, Muhammad I. Sobri, Rafidah Othman,
Fui F. Ching, Sitti Raehanah M. Shaleh

Borneo Marine Research Institute, Universiti Malaysia Sabah, Kota Kinabalu, Sabah,
Malaysia. Corresponding author: S. R. Muhamad Shaleh, [email protected]


Abstract. This study aims to investigate the effect of water temperature and stocking density in a long-
hour transportation of hybrid grouper [tiger grouper (Epinephelus fuscoguttatus) x giant grouper (E.
lanceolatus)]. A factorial design of three different water temperature levels (16, 18 and 21°C) and three
different stocking density levels (120, 180 and 240 g L
-1
) was tested in this experiment. The experiment
was conducted using a 10 L rectangular aquarium, equipped with a water chiller and aeration to maintain
the water temperature at the required level. The hybrid grouper (average body weight: 5.11±0.34 g)
were placed in each aquarium with different stocking density levels for a 12 -hour period. The results
show that the survival of fish was significantly lower (p < 0.05) at a lower water temperature level
(16°C; 50.2%), while there is no significant difference for the other temperature levels considered, 18
and 21°C. The results also show that the lowest water temperature had a significantly (p < 0.05) lower
pH value in each stocking density. The glycogen content in fish liver was significantly higher (p < 0.05)
at a low water temperature and low stocking density. A similar trend can be observed on the dissolve d
oxygen of water during the experiment. Moreover, the ammonia concentration was sig nificantly (p <
0.05) higher at a higher water temperature and a higher stocking density. The findings in the present
study suggest that hybrid grouper can be economically transported for a long hour journey at a high
stocking density (240 g L
-1
), with a water temperature level between 18 to 21°C.
Key Words: tiger grouper, giant grouper, water quality, survival rate, glycogen content.


Introduction. Hybrid grouper is a crossbreed between tiger grouper ( Epinephelus
fuscoguttatus and giant grouper (E. lanceolatus), and it was first produced in 2006 by a
group of researchers at Universiti Malaysia Sabah (Ch’ng & Senoo 2008). The hybrid
grouper has several advantages such as fast growth, high tolerance towards diseases and
extreme culture conditions and good f lesh quality, compared to the parental species
(Chor et al 2015; Anthonius et al 2018; Mohd Faudzi et al 2018; Shapawi et al 2019).
Due to these advantages, hybrid grouper had been receiving increasing attention as a
candidate for aquaculture production, especially in Southeast Asia, Taiwan and China
(Senoo 2010; Chu et al 2016; Shapawi et al 2019). As hybrid grouper is highly valued
and increase in demand, there is an increasing need to transport the juveniles or adults
from the hatcheries to farms, or to other places.
In fish farming, fish are transported from one to another place either for breeding,
stocking in nursery, or for selling. Fish transportation acts as a potential stressor that
may have a negative effect on the health and welfare of the fish ( Iversen et al 2009;
Pereira-Cardona et al 2017). Proper handling during fish transportation helps to distribute
good quality fish to other places. Appropriate procedures of fish transportation may also
expand the profitability of the business by reducing t he mortality of the fish being
transported (Gomes et al 2003). During transportation, the water quality of the holding
water may fluctuate, and eventually causes stress to the transported fish. This is because
transportation without optimum conditions may lead to metabolic disturbance, and in
turn to an increased mortality rate.

AACL Bioflux, 2021, Volume 14, Issue 2.
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As the fish are poikilotherms, changes in ambient water temperature leads to
changes in the metabolic rate (Omeji et al 2017). Most of the fish can tolerate normal
water temperature changes. However, during fish transportation, rapid changes occur in
the water’s temperature level. Due to this, prior to fish transportation, lowering the water
temperature is vital. This may reduce fish activity, metabolism as well as the bacterial
growth. Thus, this reduces the stress placed on the fish, as well as the ammonia and
carbon dioxide production in the water during transportation (Gou et al 1995; Cheng et al
2014). Other than that, water temperature plays an important role in controlling the
utilization of oxygen during the transportation of fish (Kita et al 1996; Alsop et al 1999).
Stocking density is another important factor that influences fish conditions during
the transportation. Transporting fish in high stocking density may lessen the cost by
optimising the space efficiently. On the other hand, high stocking density of fish could
also trigger a stress response through metabolic pathways (Costas et al 2008; Laiz-
Carrión et al 2012) caused by overcrowding and degradation of water quality , which
eventually results in negative biochemical and physiological modification (Bolasina et al
2006). Therefore, finding an optimal balance between maximising profit and minimising
stress response and mortality is necessary (Herrera et al 2009; Björnsson et al 2012).
Previous studies revealed that increases in stocking density during transportation led to
higher stress responses, ammonia accumulation, hydromineral imbalance and mortality
in various fish species, including tambaqui (Colossoma macropomum ) (Gomes et al
2003), winter flounder (Pseudopleuronectes americanus) (Sulikowski et al 2006), cobia
(Rachycentron canadum) (Stieglitz et al 2012), red porgy (Pagrus pagrus) (Pavlidis et al
2003) and matrinx (Brycon cephalus) (Carneiro & Urbinati 2002).
The results obtained from other grouper species may not represent the hybrid
grouper, as the tolerance of fish towards water temperature and stocking density during
fish transportation is species-dependent. Therefore, this study aims to determine the
optimum water temperature and stocking density that can be tolerated by juvenile hybrid
grouper for a long-hour transportation.

Material and Method

Experimental procedures . The experiment was conducted on February 2020 at fish
hatchery of Borneo Marine Research Institute, Universiti Malaysia Sabah (UMS), Kota
Kinabalu, Malaysia. The experimental fish were obtained from a local private farm (Batu
Payung Aquaculture) in Tawau, Sabah, Malaysia. Approximately, 300 pieces of juvenile
hybrid grouper (average body weight: 5.11±0.34 g) were bought and transported to the
fish hatchery at UMS (packing density: 65.0 -69.0 g L
-1
, temperature: 19.7-22.0°C,
dissolved oxygen: 13.0-16.7 mg L
-1
, pH: 7.04-7.02). The experimental fish were then
acclimatized for one week, and fed mari ne pellets (Leong Hup Feedmills Sdn. Bhd.,
Malaysia; crude protein: 45%, crude lipid: 8%).
A factorial design of three different levels of water temperature levels (16, 18 and
21°C) and three different stocking density levels (120, 180 and 240 g L
-1
) was tested in
this experiment. The experiment was conducted using a 10 L rectangular aquarium
placed in a water bath, equipped with a water chiller to maintain the required
temperature. Aeration was provided to facilitate the introduction of oxygen. During the
experiment, the fish (following respective stocking density) were placed in each
aquarium, and the experiment was carried out for 12 hours. The fish were observed
every hour. The water quality (temperature, dissolved oxygen and pH) was measured
using a multiparameter instrument (YSI Professional Plus handheld multiparameter, USA)
every hour during the experiment. The number of dead fish and survival rate was
recorded. At the end of experiment, the water sample in the respective treatment was
also taken for ammonia analysis. Fish (n = 3) from each treatment were sampled for
glycogen analysis.

Chemical analysis. Ammonia analysis was conducted for each treatment following the
calorimetric method proposed by Parsons et al (1984). The reading absorbance was read
at 640 μm by using a spectrophotometer (Hach DR5000TM UV -Vis Spectrophotometer,

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1100
Canada). The glycogen content was determined following the method by Montgomery
(1957). The fish samples were dissected to obtain liver tissue for analysis. The colour
intensity was read spectrophotometrically at 490 μm.

Statistical analysis. All the quantitative data expressed in percentage were subject to
arcsine transformation, prior to the statistical analysis. The quantitative data were
analysed using one-way analysis of variance (ANOVA) to determine the mean difference
among the treatment at the 0.05 significance level. The effect of temperature, packing
density and interaction between temperature and packing density were analysed using
two-way ANOVA.

Results. The survival of the fish is shown in Table 1. During the experiment, the mean
survival of fish in treatment at 18 and 21°C was 99.03 and 100% respectively. However,
at 16°C, the results were significantly lower (p < 0.05), with only half of the fish
surviving (50.20%). This shows decreasing trend as the water temperature decreased.
The results of this experiment revealed that hybrid grouper could be transported in high
stocking density by maintaining the water temperature within the range of 18 to 21°C.

Table 1
Survival (%) of juvenile hybrid grouper in different water temperature and stocking density
Mean (±SD) values (n = 3) with different superscripts within the same column are significantly difference (p <
0.05).

The glycogen content of the juvenile hybrid grouper during the experiment is presented
in Table 2. The study shows that glycogen was significantly affected (p < 0.05) by water
temperature and stocking density. The glycogen content was significantly ( p < 0.05)
lower in treatment at 21°C, compared to treatment at 16°C. Similar to that, the glycogen
content was also significantly (p < 0.05) lower with high stocking density (240 g L
-1
)
compared to with low stocking density (120 g L
-1
). A decreasing trend was observed in
terms of glycogen content when both water temperature and stocking density increa sed
from 16 to 21°C, and from 120 to 240 g L
-1
.
The water quality parameters during the experiment are shown in Table 3.
Dissolved oxygen and ammonia concentration were significantly (p < 0.05) affected by
temperature and stocking density. Meanwhile, pH of water was significantly (p < 0.05)
affected by temperature. There was a significant (p < 0.05) interaction of temperature
and stocking density on pH and ammonia concentration values in the water. The
temperature of the water corresponded to the specific temperature designed for the
experiment. The results demonstrate that a lower temperature had a significantly (p <
0.05) lower value of pH in each stocking density, compared to a higher temperature (18
and 21°C). Meanwhile, based on the results, the glycogen content significantly (p < 0.05)
decreased when the temperature increased from 16 to 21°C, and the stocking density
increased from 120 to 240 g L
-1
. A similar trend was observed on dissolved oxygen of
water during the experiment. In addition, the ammonia concentration was significantly (p
< 0.05) increased when the temperature increased from 16 to 21°C, and when the
stocking density increased from 120 to 240 g L
-1
.

Water temperature (°C) Stocking density (g L
-1
) Survival (%) Mean±SD
120 70.8
180 44.1 16
240 35.7
50.20±18.33
b
120 100
180 97.1 18
240 100
99.03±1.67
a
120 100
180 100 21
240 100
100.00±0.00
a

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Table 2
Glycogen concentration (mg/100 mg) of juvenile hybrid grouper in different water
temperature and stocking density

Mean (±SD) values (n = 3) with different superscripts within the same column are significantly difference (p <
0.05).

Table 3
Water quality parameters (temperature, dissolved oxygen, pH and ammonia
concentration) in different temperature and stocking density

Treatment Water quality
Water
temperature
(°C)
Stocking
density
(g L
-1
)
Temperature
(°C)
Dissolved
oxygen
(mg L
-1
)
pH
Ammonia
concentration
(mg L
-1
)
120 16.94±1.70
a
6.28±0.44
e
6.06±0.20
a
0.09±0.01
a
180 17.03±1.84
a
5.74±0.49
d
6.21±0.11
b
0.14±0.00
b
16
240 17.07±1.78
a
5.15±0.33
bc
6.11±0.14
ab
0.19±0.02
c
120 18.75±1.14
b
5.53±0.46
cd
7.54±0.19
d
0.15±0.00
b
180 18.60±1.16
b
5.09±0.45
b
7.40±0.18
c
0.20±0.03
c
18
240 18.58±1.08
b
4.40±0.68
a
7.42±0.18
c
0.25±0.01
d
120 21.07±0.33
c
6.26±0.50
e
7.30±0.07
c
0.21±0.01
c
180 20.93±0.40
c
5.64±0.32
d
7.35±0.06
c
0.29±0.00
e
21
240 21.00±0.35
c
4.43±0.60
a
7.30±0.09
c
0.37±0.02
f
Two-way ANOVA (P value)
Water temperature 0.00 0.00 0.00 0.00
Stocking density 0.97 0.00 0.44 0.00
Interaction 0.99 0.05 0.01 0.00
Mean (±SD) values (n = 3) with different superscripts within the same column are significantly difference (p <
0.05).

Discussion. In this experiment with different temperature and stocking density levels,
the group in the water at 16
o
C had a low survival compared to the other groups. During
the experiment, hybrid grouper in water with a lower temperature (16
o
C) showed weak
condition and activity, where the fish were less active in terms of swimming and
breathing. Based on the mean survival for each water temperature group, 18 and 21
o
C
were the optimal water temperature levels to promote a higher survival rate during fish
transportation. In general, the water temperature influences and limits the entire
behaviour and physiology of fish (Nazarudin et al 2016). It was reported that the use of a
low water temperature will decrease the metabolic rate of fish to a value lower than the
basal metabolic rate (Lili et al 2019). This might cause the respiration rate to become
very low in hybrid grouper. Other than that, previous studies on hybrid grouper also
Treatment
Water temperature (°C) Stocking density (g L
-1
)
Glycogen concentration
(mg/100 mg)
120 0.82±0.09
c
180 0.74±0.12
c
16
240 0.59±0.05
b
120 0.60±0.11
b
180 0.54±0.02
b
18
240 0.38±0.02
a
120 0.39±0.02
a
180 0.30±0.01
a
21
240 0.27±0.02
a
Two-way ANOVA (P value)
Water temperature 0.00
Stocking density 0.00
Interaction 0.36

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reported 100% mortality when the fish cultured below 14
o
C during a 30 days duration
(Zhang et al 2018). According to Cheng et al (2013), 50% of juvenile tiger grouper died
at a water temperature range of 9.8 to 12.2
o
C. It has been suggested that a low water
temperature caused slow heart movement and respiration rates (Zhang et al 2018).
The study demonstrated that stocking density and water temperature clearly
affected the water quality. The dissolved oxygen decreased as the stocking density
increased, in each water temperature group. In line with this study, dissolved oxygen
levels were found to be lower at a higher stocking density in orange-spotted grouper
during the transportation period (Xavier et al 2018). In all ages of hatchery-reared
grouper, the dissolved oxygen dropped significantly when increasing the stocking density
from 50 to 200 fish L
-1
, during simulated transportation (Estudillo & Duray 2003). This is
primarily due to the higher oxygen consumption in the higher stocking density of fish.
Chatterjee et al (2010) reported that stocking density greatly influenced the oxygen
consumption during fish transportation. Moreover, fish need a large amount of oxygen in
stressful conditions in order to counteract their excitement in high stocking density
(Xavier et al 2018). Meanwhile, increasing the water temperature also led to an increase
in fish respiration, and consequently a 3-5 time increase in oxygen consumption by fish
(Lili et al 2019). Dissolved oxygen was decreased when the water temperature increased
in this study. In agreement with this result, simulated transportation of hatchery-reared
grouper showed that the dissolved oxygen decreased when the water temperature
increased from 23 to 29
o
C in different age groups (35, 45 and 60 days old) (Estudillo &
Duray 2003).
Generally, increasing the water temperature contributed to a reduced pH va lue
(Xavier et al 2018). This is because the rising water temperature commonly causes an
increased respiration rate, which leads to the excretion of carbon dioxide in the water. In
a previous study, an increase in water temperature during the transportation of juvenile
goldfish led to an increase in the pH value of the water (Lili et al 2019). Excess levels of
carbon dioxide may result in hypercapnia (high carbon dioxide concentration in blood)
and acidosis, which consequently causes a higher mortality rate (Harmon 2009).
However, this study shows that the lowest water temperature resulted in a low pH value
in the water. In this study, the opened system was used, whereby the aquariums were
filled with water and supplied with aeration continuously from an external source. Thus,
accumulated carbon dioxide in the water caused by respiration process did not occur, and
the pH was preserved, with the exception of treatment with a low water temperature.
Acidic water with a low temperature might be caused by the higher bacteria number, due
to fish mortality. The production of carbon dioxide was not only by the respiration of fish,
but was also contributed by the bacteria contained in the water.
Ammonia is the main nitrogenous product that is excreted by teleosts (de Oliveira
et al 2008; Franklin & Edward 2019). The bulk of excretion is discharged from the blood,
which is then excreted through the gills (Franklin & Edward 2019). The ammonia toxicity
to fish depends on the concentration of unionized ammonia (NH 3) (de Oliveira et al 2008)
in the water. In this study, the ammonia concentration increased when the stocking
density and water temperature increased. However, the ammonia concentration was
within the tolerable limit of the hybrid grouper, and did not cause fish mortality. Increase
the stocking density and water temperature increased energy demand, which led to the
direct deamination process of important tissue energy sources for energy production,
resulting in an increased ammonia level (Philip & Rajasree 1996; Sm utna et al 2002;
Xavier et al 2018). A previous study on orange spotted grouper also showed similar
findings. The ammonia concentration increased as stocking density increased from 20 to
50 fish L
-1
during fish transportation (Xavier et al 2018). Similarly, Estudillo & Duray
(2003) reported that the ammonia concentration increased when 35, 45 and 60 days old
hatchery-reared grouper larvae underwent simulated transportation at different stocking
density levels. Estudillo & Duray (2003) also reported that an increase in water
temperature during the experiment contributed to the increase in ammonia concentration
when transporting 35, 45 and 60 days old hatchery-reared grouper larvae.
Glucose, which plays a central role in providing energy for metabolism, is
primarily stored in the liver as glycogen (Chang et al 2007). Glycogen metabolism is the

AACL Bioflux, 2021, Volume 14, Issue 2.
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1103
principal energy source in both vertebrates and invertebrates, and is influenced by
environmental fluctuation (Bacca et al 2005; Chang et al 2007). In this study, the
glycogen concentration in hybrid grouper decreased when the stocking density in each
water temperature group increased. It is also showed a similar trend when the water
temperature increased for each stocking density. Increasing the stocking density and
temperature was reported to increase the demand for energy and metabolism during fish
transportation (Liu et al 2016; Nazarudin et al 2016). Other than that, an increase in
stocking density and water temperature also led to stressful conditions (Manuel et al
2014; Wu et al 2020). Thus, the stress conditions produce a series of defense
mechanisms that need more energy to be used compared to normal conditions (Lupatsch
et al 2010; Liu et al 2016).

Conclusions. The findings by this study suggest that hybrid grouper can be transported
at extremely high stocking density levels (240 g L
-1
) compared to other grouper species
(120-150 g L
-1
) at temperature of 18 to 21
o
C with higher survival and less stress. Hence,
the cost of long-hour transportation for hybrid grouper can be minimized and contributed
in increased the profit for the fish farmer.

Acknowledgements . This study was funded by UMS Fund Research Grant Scheme
(SDK0057-2018) from Universiti Malaysia Sabah, Malaysia. The authors would like to
thank the staff members and research assistants at the Marine Fish Hatchery, Borneo
Marine Research Institute, Universiti Malaysia Sabah, for their technical assistance
throughout this study.

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AACL Bioflux, 2021, Volume 14, Issue 2.
http://www.bioflux.com.ro/aacl
1106
Received: 07 January 2021. Accepted: 20 March 2021. Published online: 29 April 2021.
Authors:
Norfazreena Mohd Faudzi, Borneo Marine Research Institute, Universiti Malaysia Sabah, Jalan UMS, 884 50, Kota
Kinabalu, Sabah, Malaysia, e-mail: [email protected]
Muhammad ’Izzat Sobri, Borneo Marine Research Institute, Universiti Malaysia Sabah, Jalan UMS, 88450 , Kota
Kinabalu, Sabah, Malaysia, e-mail: [email protected]
Rafidah Othman, Borneo Marine Research Institute, Universiti Malaysia Sabah, Jalan UMS, 88450, Kota
Kinabalu, Sabah, Malaysia, e-mail: [email protected]
Fui Fui Ching, Borneo Marine Research Institute, Universiti Malaysia Sabah, Jalan UMS, 88450, Kota Kinabalu,
Sabah, Malaysia, e-mail: [email protected]
Sitti Raehanah Muhamad Shaleh, Borneo Marine Research Institute, Universiti Malaysia Sabah, Jalan UM S,
88450, Kota Kinabalu, Sabah, Malaysia, e-mail: [email protected]
This is an open-access article distributed under the terms of the Creative Commons Attribution License, which
permits unrestricted use, distribution and reproduction in any medium, provided the original author and source
are credited.
How to cite this article:
Faudzi N. M., Sobri M. I., Othman R., Ching F. F., Shaleh S. R. M., 2021 Water temperature and stocking
density for long-hour transportation of hybrid grouper Epinephelus fuscoguttatus x E. lanceolatus. AACL Bioflux
14(2):1098-1106.