Egyptian Journal of Aquatic Research 47 (2021) 329–334

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Egyptian Journal of Aquatic Research

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Effect of varying dietary protein levels on growth performance and

survival of milkfish Chanos chanos fingerlings reared in brackish water
pond ecosystem q
Muneer Hussain a, Habib Ul Hassan a,b,⇑, Mohammad Abdul Momin Siddique c, Khalid Mahmood a,
M.F.A. Abdel-Aziz d, Muhammad Y. Laghari e, Naveed A. Abro a, Karim Gabol b, Nisar f, Saba Rizwan g,
Halima h
a
Fisheries Development Board, Ministry of National Food Security and Research, Pakistan
b
Department of Zoology (MRCC), University of Karachi, Karachi 75270, Pakistan
c
Department of Oceanography, Noakhali Science and Technology University, Noakhali 3814, Bangladesh
d
Aquaculture Division, National Institute of Oceanography and Fisheries (NIOF), Egypt
e
Department of Fresh Water Biology & Fisheries, University Of Sindh, Jamshoro, Pakistan
f
Department of Environmental Sciences, SMIU, Karachi, Pakistan
g
Jinnah University for Women, Nazimabad, Karachi 74600, Pakistan
h
KIBGE, University of Karachi, Pakistan

a r t i c l e i n f o a b s t r a c t

Article history: The present study investigated the effects of different dietary protein levels on the growth and survival of
Received 21 January 2021 milkfish Chanos chanos for 90 days feeding trial. Milkfish with an average body weight of 10.81 ± 0.07 g
Revised 7 May 2021 were collected from the coastal zone using a manual dragnet. The study tested four diets, 10% (T1), 15%
Accepted 10 May 2021
(T2), 30% (T3) and 38% (T4) of proteins containing feed with triplicates. These diets were offered to the
Available online 2 June 2021
milkfish juveniles in triplicate earthen ponds (1.5 ha). Juveniles were stocked at 10,000 ind./1.5 ha,
and initially feeding rate of 10% biomass per day. The feeding rate adjusted according to fish biomass
Keywords:
on a weekly basis. The present results showed that fishes fed 30% of proteins-containing feed have the
Growth performance
Survival
highest weight gain (WG), specific growth rate (SGR), average daily weight gain (ADWG), survival, and
Protein requirements the best feed conversion ratio (FCR) compared to the other treatments. The present study suggested that
Brackish water 30% crude protein is the optimum level for the best growth performance and for obtaining a high yield of
Pond culture milkfish C. chanos reared in the brackish water ponds.
Ó 2021 National Institute of Oceanography and Fisheries. Hosting 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/).

Introduction is the rapidly growing food production sector and has been contin-
uously increasing over the last three decades (Azim et al., 2003;
Fish is one of the primary sources of high-quality protein to be Ahmad et al., 2020, 2021). The development of the aquaculture
consumed by one billion people worldwide (Ahmad et al., 2020, industry and its success necessitate knowledge of each fish species’
2021; Khalid et al., 2020). Aquaculture plays a role in preserving nutritional requirements under different environmental condi-
the wild fishes and maintaining food and nutritional reliability tions. Therefore, determining the optimum dietary protein levels
(Ntsama et al., 2018; Kumari et al., 2018; Hassan et al., 2021a, is essential to obtain the best growth, water quality maintenance,
2021b). Moreover, the utilization of marine water and large coastal and economic efficiency (NRC, 2011).
areas can meet the requirements of a vast population of animal A protein containing diet is considered a source of non-essential
proteins (Habib et al., 2020). Globally, mariculture, or aquaculture amino acids, essential amino acids, energy for biological activities,
and a tool for maintenance and tissue repair (Zehra and Khan,
2012; Li et al., 2016). The dietary protein requirement is affected
Peer review under responsibility of National Institute of Oceanography and
Fisheries. by several factors, including species, age/size of the fish, dietary
⇑ Corresponding author at: Department of Zoology (MRCC), University of Karachi, protein quality, energy level and the protein to energy balance
Karachi 75270, Pakistan. (Teshima et al. 1984). Milkfish Chanos chanos belong to the family
E-mail addresses: [email protected], [email protected] (H.U. Chanidae, and it is an economically important brackish water fish
Hassan).

https://doi.org/10.1016/j.ejar.2021.05.001
1687-4285/Ó 2021 National Institute of Oceanography and Fisheries. Hosting 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/).
M. Hussain, Habib Ul Hassan, Mohammad Abdul Momin Siddique et al. Egyptian Journal of Aquatic Research 47 (2021) 329–334

species cultured in Southeast Asia. Milkfish is a suitable candidate as dissolved oxygen (DO), pH, and temperature were observed
for aquaculture for their fast growth, efficient use of natural feeds, daily using Multi-parameter (Hanna HI9829). The pond water
herbivore feeding habit, resistance to disease, and tolerance to a salinity was measured by a hand-held refractometer (RHS-
wide range of ecological factors (Jana et al., 2006). 10ATC) in the early morning. Alkalinity, ammonia, nitrite and
In addition, milkfish is a euryhaline species and can thrive in nitrate were analyzed on a daily basis (APHA, 1995). For pond
waters from 10 to 32 PSU salinity (Froese and Pauly, 2021). This management, ponds were cleaned bi-weekly and exchanged 30%
species has been cultured on a large scale in many countries like of water depending on water quality. A continuous inspection of
Indonesia, Taiwan, and the Philippines (Vasava et al., 2018). Many the screen net was installed at the inlet and outlet gates of the
studies have been conducted dealing with the nutritional require- pond and maintenance of pond dykes. About 4–7 cm of bottom soil
ments for the growth performance of C. chanos (Mwangamilo and was removed from each pond to eliminate different heavy metals,
Jiddawi, 2003; Jana et al., 2006; Vasava et al., 2018). The nutritional toxins and the accumulated feed stored at the bottom, hurrying
requirements of fishes usually differ due to many factors such as organic matter decay and oxidation of abridged amalgams. Liming
fish size, rearing system, environmental conditions, availability of was carried out to nullify the soil’s acidity and increase the overall
natural food, feeding rate, feed ingredients and feeding time. alkalinity and total hardness concentration that enhance the pri-
Therefore, the present work aims to examine the effects of various mary productivity in the pond. The organic fertilizer was used
dietary protein levels on the growth performance and survival of C. before filling with water to increase the abundance of zooplankton
chanos reared in the brackish water ponds. in the ponds. Inorganic nitrogen and phosphorous fertilizers were
applied to stimulate phytoplankton to increase primary productiv-
ity and to influence the zooplankton growth. Dolomite was used to
Materials and methods
help in the plankton development (Fig. 1).
Ethical statement
Growth parameters
This study was carried out with the strict commendations and
approval of the Ethical Committee for Experiments on Aquaculture The growth parameters were calculated according to the fol-
of the FDB, MNFS & R, Pakistan. lowing formula:
WG; g ¼ A - B
Experimental fishes and acclimatization
where A = final body weight and B = Initial body weight
The trial was conducted in earthen ponds at Reliance Aqua SGR; %=day ¼ ½ðln FBW  ln IBW Þ=day
100
Farm, located along the Khraro Creek (N: 24.24546766, E:
where: Ln = Natural logarithm,
67.460967) coastal region of Sindh, Pakistan. Milkfish juveniles
FBW = final body weight,
were collected from the wild sources near Samoo and Jat village
IBW = initial body weight.
District Thatta, Sindh. Samples of C. chanos were caught by drag-
nets. Juveniles were transported to the culture site in aerated SRð%Þ ¼ A=B  100
water tanks and stocked in brackish water ponds (area of 1.5 ha,
depth of 4 ft). The initial stocking density of the juveniles was where A = No. of fish harvested and B = No. of fish stocked
10,000 ind./1.5 ha. Before stocking into the pond, juveniles were FCR = Feed given (dry weight) (g) / Body weight gain (wet
slowly acclimatized to the water of the experimental ponds for weight) (g)
one hour. For acclimatization, pond water was slowly mixed with ADWG g/day = Final weight (g) – Initial weight (g) / days.
the water of the individual stocking bucket. Juveniles with an aver-
age initial weight of 10.81 ± 0.07 g were uniformly distributed Chemical analysis of feed
(10,000 ind./hectare) in 4
3 = 12 ponds. A total of four formulated
feeds with different protein levels (10% (T1), 15% (T2), CP 30% (T3) The biochemical analysis of feed was performed according to
and 38% (T4)) were fed to the juveniles for 90 days. AOAC (2000). The gross energy (GE) was estimated to formulate
the diets. The factors were 23.62, 39.5 and 17.56 KJ/g for crude pro-
tein (CP), ether extract (EE) and carbohydrates, respectively, were
2.3. Feed formulation and feeding
used for this purpose (NRC, 1993).
The feed was formulated with different crude protein levels, as
shown in Table 1. The formulation of feed was done based on Pear- Statistical analysis
son’s square method. The ingredients used in the experimental
diets were prepared with fish meal (FM), soybean meal, maize, A one-way ANOVA was performed to estimate the differences
mustard oil cake, rapeseed cake and rice polish. Mustard oil cake among the treatments, and when a significant variation was
and rapeseed cake were extracted by expeller-pressing, whereas detected, a subsequent analysis of Tukey’s multiple range test
seeds were heated by steam before oil extraction. All the feed was performed. A significant level for all analysis was set for
ingredients were obtained from the local market. These ingredients p < 0.05. All the statistical analysis was performed using SPSS
were ground, mixed and then pelleted (2 mm) by a small manufac- (v18) statistical software and Microsoft Excel 2010.
tory unit near the experimental ponds. Pellets were dried using a
hot-air drier and stored at 20 °C. All diets were prepared almost Results
to contain 25% fish meal protein and 75% plant protein. The feed
was given to fish at 10% of body weight as a feeding rate that Water quality
was further adjusted according to the body weight using (KERN
digital balance), fish fed four times a day (6 am, 10 am, 2 pm, Physicochemical parameters significantly differed among the
and 6 pm). treatments (p < 0.05) (Table 2). Mean values of DO, temperature,
Feeding was done manually to ensure ingestion of feed entirely pH, salinity, nitrite, nitrate, ammonia and total alkalinity ranged
by the fish. The water quality parameters of the culture pond, such between 5.66 and 6.25 mg/L, 28.57–29.1 °C, 8.05–8.2, 24.89–
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M. Hussain, Habib Ul Hassan, Mohammad Abdul Momin Siddique et al. Egyptian Journal of Aquatic Research 47 (2021) 329–334

Table 1
Biochemical analysis and nutritional composition of the pelleted feed used as food for the milkfish during the experiments. (g/100 g dry diet).

Feed ingredients The protein level in experimental diets

10% (T1) 15% (T2) 30% (T3) 38% (T4)
Rice polish (g/ 100 g) 80 81 32 11
Fish meal (g/ 100 g) 5 6.5 13.5 17
Soybean meal (g/ 100 g) – 7.5 22.5 40
Maize (g/ 100 g) – 5 5 5
Mustard oil cake (g/ 100 g) – – 15 15
Rapeseed cake (g/ 100 g) – – 12 12
Starch (g/ 100 g) 15 – – –
Total (g/ 100 g) 100 100 100 100
Chemical composition based on dry matter
Moisture (%) 8.4 8.19 7.89 8.59
Crude Protein (%) 10.8 15.84 30.10 38.36
Crude fat (%) 7.08 7.73 7.05 5.89
Ash (%) 13.55 15.16 10.65 9.15
Crude fiber (%) 17.92 18.45 9.92 5.56
NFE* 50.62 43.67 41.35 40.20
Gross energy, Kjg1** 17.37 17.69 18.88 19.43

Note: * Nitrogen free extract was calculated by difference:  100  (Protein + fat + fiber + Ash).
**was calculated according to NRC (1993).

Fig. 1. Design of the earthen ponds.

Table 2
The water quality parameters in different treatments of milkfish (C. chanos). Data are presented as the mean ± SE.

Hydrological parameters Treatment ponds

T1 T2 T3 T4
Dissolved oxygen (mg/L) 5.71 ± 1.12 6.25 ± 0.53 5.66 ± 1.11 5.72 ± 1.40
Temperature (°C) 28.57 ± 0.13 28.77 ± 0.28 28.9 ± 0.70 29.1 ± 0.80
pH 8.05 ± 0.52 8.1 ± 0.34 8.2 ± 0.20 8.09 ± 0.40
Salinity (ppt) 24.92 ± 0.48 25.23 ± 0.44 25.40 ± 0.4 24.89 ± 0.70
Nitrite (mg/L) 0.04 ± 0.01 0.03 ± 0.01 0.04 ± 0.00 0.04 ± 0.01
Nitrate (mg/L) 2.81 ± 0.05 2.62 ± 0.08 2.81 ± 0.03 2.83 ± 0.05
Ammonia (mg/L) 0.08 ± 0.01 0.09 ± 0.02 0.09 ± 0.01 0.08 ± 0.02
Total Alkalinity (mg/L) 152.64 ± 2.12 153.5 ± 2.23 152.11 ± 2.14 153 ± 1.54

25.40 ppt, 0.03–0.04 mg/L, 2.62–2.83 mg/L, 0.08–0.09 mg/L and Growth parameters
152.11–153.5 mg/L, respectively.
Growth response, feed utilization and survival of milkfish C.
chanos after 90 days are presented in Table 3. The statistical anal-

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M. Hussain, Habib Ul Hassan, Mohammad Abdul Momin Siddique et al. Egyptian Journal of Aquatic Research 47 (2021) 329–334

Table 3
Growth response and feed utilization of milkfish C. chanos feeding on experimental diets containing different levels of protein.

Growth parameters Treatments

T1 T2 T2 T4
Initial body weight 10.81 ± 0.07 10.0 ± 0.08 10.40 ± 0.2 10.40 ± 0.2
Final body weight 190.30 ± 0.06c 220 ± 0.06b 275 ± 0.06a 272 ± 0.06ab
Weight gain (g) 179.89 ± 0.01c 210 ± 0.02b 264.6 ± 0.04a 261.6 ± 0.04ab
Feed conversion ratio 2.5 ± 0.001c 2.1 ± 0.001c 1.10 ± 0.001a 1.14 ± 0.002b
Specific growth rate (%) 2.60 ± 0.02b 2.83 ± 0.01ab 3.05 ± 0.02a 3.00 ± 0.01a
Average daily weight gain (g/day) 1.99 ± 0.05c 2.30 ± 0.073b 2.93 ± 0.06a 2.90 ± 0.06a
Survival (%) 71c 83b 92a 91a

Note: The mean ± SD of (triplicate) treatments in the same row with a number of different superscripts differs significantly between them (p < 0.05).

ysis did not show any significant differences among the treatments to decline, as it was observed with 38% of CP (T4). In the same con-
in initial body weight (p > 0.05). The growth performance was sig- text, Ahmad et al. (2019), Romanova et al. (2018) and Bureau
nificantly changed by different dietary protein levels (p < 0.05). The (2002) suggested that the increase of dietary protein levels
growth parameters were significantly higher in T3 compared to the resulted in a decline in FBW, WG, SGR and low FCR. Several previ-
other treatments. The values of FBW, WG, ADWG and SGR were ous studies have shown that the optimum dietary protein levels in
275 g, 264.6 g, 2.93 g and 3.05%/ day with fish fed dietary 30% of the diet tend to cause a reduction in the growth performance of
CP (T3), the subsequent treatment was occupied by the fish fed fish (Du et al., 2005; Balito-Liboon et al., 2018; Sukardi et al.,
dietary 38% of CP (T4), which recorded 272 g, 261.6 g, 2.90 g and 2019; Roy et al., 2020). The dietary protein requirement for milk-
3.0%/day of FBW, WG, ADWG and SGR, respectively. Insufficient fish is between 30 and 40% (Pascual, 1988; Coloso et al., 1987).
amounts of protein (T1- 10% of CP and T2- 15% of CP) in the diet Likewise, a previous study emerged that the optimum dietary
showed reducing the growth performance of fish. protein of milkfish with an average initial weight of 0.25 to
4.43 g was 40% CP regardless of the protein sources and rearing
Feed conversion ratio place (Jana et al., 2006). However, the range of dietary 30% to
40% of CP is very wide. Therefore, many factors control the deter-
The FCR value significantly varied among the groups (p < 0.05, mination of the optimum dietary protein with high accuracy as
Table 2). Where T3 had the best FCR (1.10) followed by T4 (1.14) species, age, feeding regime, protein quality, presence of neutral
and T2 (2.1), while the worst FCR was achieved by T1 (2.5), food, environmental conditions and stoking rate. The protein
whereas 10% or 15% CP in the milkfish diet was not fulfilled the requirement of fish increased during an early age than the consid-
protein requirement. erable age in the same species (Mwangamilo and Jiddawi, 2003).
Further, it has been noticed that the diet containing 32% of CP
Survival rate achieved the best growth of milkfish during their initial weight
of 12 to 15 g. Pascual (1988) mentioned that the milkfish with
The survival rate was significantly affected (p < 0.05) by the an average initial weight of 0.5 to 0.8 g required 40% CP in their
dietary protein levels; the highest survival was 98% and was diet.
obtained in T3 followed by T4 and T2 while T1 had the least sur- In the current study, milkfish juveniles fed four times daily. It
vival that was 70%. No significant differences were detected has been reported that feeding frequency affects the dietary pro-
between T3 and T4 in the survival rate, but they were significantly tein level requirements (Fan et al., 2016). The protease enzyme
higher than T2 and T1, respectively. activities increased with fish fed four times than those fed two
times. Thus, the fish are more able to utilize dietary fat and carbo-
hydrate content with increased feeding frequency. On the other
Discussion
hand, the experimental diets in this study contained 75% of plant
proteins. The diets of T3, T4 contained mustard oil cake and rape-
In the present study, temperature, salinity, dissolved oxygen,
seed cake. Milkfish is herbivore species and more resistant to con-
pH, and other water quality parameters were within the tolerable
sume high content of glucosinolate in the diet compared to the
ranges for C. chanos (Jana et al., 2006). Mwangamilo and Jiddawi
carnivore species (Hardy, 2010). Rapeseed may have less amount
(2003) reported that water quality parameters did not differ
of lysine but contain higher amount of methionine. Mustard oil
among the groups that fed other dietary proteins, which indicated
cake is rich in both lysine and sulphuric acid than soybean (Devi
that they remained below the critical levels. The concentrations of
and Devi, 2011). The relationship between the feed ingredients
ammonia in the rearing water increased with an increased level of
and the protein levels was studied (Teshima et al., 1984).
proteins in the diets (Hassan et al., 2021b).
Teshima et al. (1984) reported that the best growth of milkfish
In the present study, the protein levels in the diets had signifi-
fed a purified diet containing 35% casein and 15% gelatins as pro-
cant effects on growth indicators and feed efficiency of the milkfish
tein sources.
C. chanos. This agreed with similar data reported for C. chanos
The rearing condition and culture systems have a significant
(Sukardi et al., 2019). An increase of dietary protein levels in the
role in determining the optimum protein level of milkfish. Natural
diets had positively affected the growth performance of C. chanos.
food is an additional source of essential amino acids, highly unsat-
On the contrary, the levels of dietary protein levels did not affect
urated fatty acids (HUFA), and ascorbic acids. The optimal dietary
the growth of the Acanthopagrus Arabicus (Ahmad et al., 2019)
proteins for the best growth of milkfish were recorded 35% CP
and the Blackfin seabream Acanthopagrus berda (Rahim et al.,
reared in the floating net cages (Suresh et al., 2018).
2016). Our results emphasized that the milkfish juveniles fed a diet
In the current study, an SGR model plotted against the increased
containing 30% of CP had the best FBW, WG, ADWG, SGR, and FCR
protein level by second-order polynomial regression of fish growth
compared to 10 or 15% of CP containing feed.
(y = 2E.05
3 + 0.0029
2 – 0.1063 + 1.7994; R2 = 1; Fig. 2) which
Furthermore, the growth rate increased with increasing the
revealed a reduced growth rate at low and high protein regimes.
dietary protein level up to the limit, then the growth curve begins
332
M. Hussain, Habib Ul Hassan, Mohammad Abdul Momin Siddique et al. Egyptian Journal of Aquatic Research 47 (2021) 329–334

Fig. 2. Polynomial regression between specific growth rate (SGR%/d) and protein levels showing, 30% crude protein is the optimum dietary protein level for the maximum
growth of C. chanos.

According to polynomial regression, the optimum CP was 30% Declaration of Competing Interest
under the experimental conditions. The growth curve reduced with
dietary protein to 38%. This corroborates that protease activity The authors declare that they have no known competing finan-
raised with increasing the dietary protein levels to the maximum, cial interests or personal relationships that could have appeared
while this activity reduced at high dietary protein levels (Jana et al., to influence the work reported in this paper.
2006). The excess protein level in fish diet could be wasted or
excreted with feces and converted to energy (Daudpota et al., Acknowledgements
2014). As well, the present results cleared that the survival rate
of milkfish was excellent under all dietary protein levels tested. The authors express their thankfulness to the Fisheries Devel-
However, the statistical analysis showed significant differences opment Board, MNFS & R, for the financial sustenance delivered
between the survival rates among treatments, increasing the diet to carry out the study. We are grateful to the Marine Reference Col-
protein up to the optimum level (30% CP). lection and Resource Center, Zoology, UOK, for laboratory space
and logistics.

Conclusion
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