Animal Feed Science and Technology 269 (2020) 114685

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Animal Feed Science and Technology

journal homepage: www.elsevier.com/locate/anifeedsci

Influence of graded level of dietary protein with equated level of

limiting amino acids on growth, feed utilization, body indices and
nutritive profile of snubnose pompano, Trachinotus blochii
(Lacepede, 1801) reared in low saline water
D Linga Prabu a, *, Sanal Ebeneezar a, S Chandrasekar a, C.S. Tejpal b, M. Kavitha a,
P Sayooj a, P. Vijayagopal a
a
ICAR-Central Marine Fisheries Research Institute, Post Box No: 1603, Ernakulam North (PO), Kochi, 682018, Kerala, India
b
ICAR-Central Institute of Fisheries Technology, CIFT Road Matsyapuri, Willingdon Island, Kochi, Kerala, 682029, India

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

Keywords: An experiment was conducted to delineate the effect of varying level of dietary protein with
Snubnose pompano balanced level of limiting amino acids such as lysine and methionine on growth, feed utilization,
Mariculture body indices and digestive enzymology of snubnose pompano, Trachinotus blochii reared in low
Body indices
saline water. There were four experimental diets formulated with 350 (CP350), 400 (CP400), 450
Growth rate on metabolic body weight
(CP450) and 500 g/kg (CP500) crude protein and each with 100 g/kg crude fat and were assigned
Daily growth coefficient
Protein requirement to four treatments, CP350, CP400, CP450 and CP500 respectively. Totally eighty four fingerlings
with the body weight in the range of 16− 18 g were randomly distributed in four treatment groups
with each of three replicates. Fish were fed to apparent satiation twice daily with the assigned
experimental diet. At the end of 13 weeks of feeding trial, weight gain percentage (WG%) and
specific growth rate (SGR) of fish fed with CP400 and CP450 diets were significantly higher than
those of fish fed with CP350 and CP500 diets. Growth rate on metabolic body weight (GRMBW),
daily growth coefficient (DGC) and average daily growth (ADG) were higher in CP400 group and
lower in CP350 group. The viscerosomatic index (VSI), hepatosomatic index (HSI), gastrosomatic
index (GSI) and intra-peritoneal fat ratio (IPFR) showed an increasing trend as the crude protein
content of the experimental diet increases and the maximum value was witnessed in CP500
treatment. Crude lipid and protein content of whole body tissue was found highest in CP400 and

Abbreviations: pNPP, p-nitrophenyl palmitate; ADG, average daily growth; ALT, alanine aminotransferase; ANOVA, analysis of variance; AST,
aspartate aminotransferase; BG, biomass gain; CI, cephalic index; CP, crude protein; CP350, crude protein 350 g/kg; CP400, crude protein 400 g/kg;
CP450, crude protein 450 g/kg; CP500, crude protein 500 g/kg; DE, digestible energy; DGC, daily growth coefficient; DM, dry matter; DMRT,
Duncan’s Multiple Range Test; DP/DE, digestible protein to digestible energy; EAA, essential amino acid; FCR, feed conversion ratio; FGR, feed gain
ratio; KMnO4, potassium permanganate; FI, feed intake; FIABS, absolute feed intake; FIMBW, feed intake per metabolic body weight; FIPCT, feed intake
of fish on % of body weight; FY, fillet yield; GRMBW, growth rate on metabolic body weight; GSI, gastrosomatic index; HPLC, high-performance
liquid chromatography; HIS, hepatosomatic index; IPFR, intra-peritoneal fat ratio; MBWG, mean metabolic bodyweight; MY-HL, meat yield-
headless; MY-HO, meat yield head-on; NEAA, non-essential amino acid; NFE, nitrogen free extract; PER, protein efficiency ratio; PPV, protein
productive value; SEM, standard error of the mean; SGR, specific growth rate; VSI, viscerosomatic index; Wf, mean final weight; WG, geometric
mean body weight; WG%, weight gain percentage; WG, weight gain; Wi, mean initial weight.
* Corresponding author at: Post Box No: 1603, Marine Biotechnology Division, ICAR-Central Marine Fisheries Research Institute, Ernakulam
North (PO), Kochi, 682018, Kerala, India.
E-mail address: growelprabu@gmail.com (D.L. Prabu).

https://doi.org/10.1016/j.anifeedsci.2020.114685
Received 9 January 2019; Received in revised form 6 March 2020; Accepted 19 September 2020
Available online 28 September 2020
0377-8401/© 2020 Elsevier B.V. All rights reserved.
D.L. Prabu et al. Animal Feed Science and Technology 269 (2020) 114685

lowest level was noticed in CP350 group. The maximum intestinal protease activity was noticed
in CP400 group. In conclusion, analysis of second degree polynomial regression revealed that the
optimum dietary protein level could be in the range of 395.3–426.7 g/kg for maximum growth of
juvenile snubnose pompano.

1. Introduction

The snubnose pompano (Trachinotus blochii) also known as silver pompano is one of the high value tropical marine fishes that is
being used for mariculture in Indo-Pacific region countries like China, Vietnam, Malaysia, India and the Philippines due to certain
adorable attributes such as seed availability, faster growth rate, tolerance to wide range of salinity, lack of bones in the meat, better
meat flavour and quality (Gopakumar et al., 2012). Globally, snubnose pompano production reached about 0.11 million mt during
2014 (FAO, 2016-2018) with major chunk from China through net cage farming, low saline pond culture and re-circulatory aqua­
culture system. Mariculture of snubnose pompano is presently done in offshore sea cages, brackishwater and backwater cages as well as
coastal ponds in China, Indonesia, India, Philippines, Taiwan, Thailand and Vietnam. Offshore sea cage farming of snubnose pompano
in commercial scale has been practiced in Vietnam.
Culture of snubnose pompanois a new avenue for mariculture practices after its success in seed production technology (Gopakumar
et al., 2012). Being a euryhaline fish species it can tolerate wide range of salinity and temperature from 0 to 65 g/L and 25–29 ◦ C
respectively (Gopakumar et al., 2011). The permissiveness to salinity is an added advantage in the sense that it can be cultured even in
coastal low saline waters (Gopakumar et al., 2012), and inland low and high saline waters by aquaculture farmers those who lack
access to coastal waters. Asian countries are having great potential for culture of snubnose pompano in offshore and coastal sea cages
as well as pond culture in coastal and inland saline soil areas which may not be suitable for any agriculture activity.
Like any aquaculture operation, feed is an important and limiting component in pompano aquaculture operation. Globally, culture
of snubnose pompano is being practiced with high level of protein and fat content in diets. Protein is the most expensive component in
the diet of any fish, hence dietary protein levels directly impinge on production cost. Most marine fish diets contain high level of
protein because carnivorous species have higher dietary protein requirements (NRC, 2011). Protein being the most indispensable
nutrient of fish feeds (Wilson, 2002), several studies have been conducted to explicate the optimum dietary protein requirement for
marine fish species to develop more cost-effective, nutritionally balanced practical diets. The fishes fed with excess of dietary protein
than the non-protein energy may limit the growth and also wasteful (Hossain et al., 1998). Therefore, balancing with non-protein
energy sources such as carbohydrates and lipid may have protein sparing action if the protein is available as optimum or slightly
suboptimum level but should be balanced with amino acid composition to ensure adequate growth. Similarly, insufficient level of
dietary protein cause retardation of growth, loss of body weight and emaciation due to removal of protein from less vital organs
particularly fish muscle in order to maintain the functions of more vital organs (Monentcham et al., 2009). Protein as such may not be
required for fish growth but it needs mixture of indispensable and dispensable amino acids in a balanced manner to synthesize new
proteins or maintain existing proteins in tissues (Wilson and Halver, 1986).
Currently, little is known about the optimum dietary protein level for snubnose pompano for the development of species specific
nutritionally balanced feed formulation which is not available for snubnose pompano that creates an obstacle for commercial pro­
duction.Therefore, the present research focuses to determine the effect of graded level of dietary protein in snubnose pompano,
Trachinotus blochii with respect to growth, feed conversion ratio, nitrogen retention and carcass composition with balanced level of
limiting amino acids, lysine and methionine in low saline waters under controlled experimental conditions.

2. Material and methods

2.1. Experimental animal

Snubnose pompano fries with an average weight of 1.5–2.0 g size were procured from ICAR-Mandapam Regional Centre of Central
Marine Fisheries Research Institute, Mandapam, India to Wet Laboratory of ICAR-Central Marine Fisheries Research Institute, Kochi,
India. The fishes were stocked in 3 FRP tanks of 1 ton capacity each with round the clock aeration and bio-filter fixed at the centre of
the tank. In order to ameliorate the handling stress the fishes were given mild dose of potassium permanganate (KMnO4) dip treatment
for 2 min in next day morning. Then vitamin C was also given to the fishes as bath treatment. The fishes were initially stocked in 20 g/
kg salinity sea water and gradually acclimatized to 5 g/kg salinity at the end of the acclimation period of 60 days. The fishes were fed
with a diet containing 450 g/kg crude protein and 100 g/kg crude fat during the acclimation period and 95 % of survival was achieved.
The fishes were grown up to an average size of 16− 20 g and selected for the present experiment.

2.2. Experimental design

The experiment was conducted for a period of 13 weeks on 12 numbers of square glass aquarium (60 × 60 × 50 cm, 180 L capacity)
covered with perforated mosquito nets to avoid leaping of fishes. The tanks were affixed with black colour stickers on the sides to avoid
too much light and external disturbances. The tanks were initially washed and filled with potassium permanganate solution (4 mg/L)
and were left overnight. Then they were flushed out in the very next day and were thoroughly washed with clean water. Then the tanks

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D.L. Prabu et al. Animal Feed Science and Technology 269 (2020) 114685

were filled with saline water of 5 g/kg. Totally eighty four fingerlings with an average body of 16− 18 g were randomly distributed in
four treatment groups such as CP350, CP400, CP450 and CP500 with each of three replicates and hence each tank contains seven
fishes. The total volume of water in each tank was maintained about 160 L throughout the experimental period.

2.3. Experimental feed preparation

There were four experimental diets formulated with 350, 400, 450 and 500 g/kg crude protein and each with 100 g/kg crude fat
and named as diet CP350, CP400, CP450 and CP500 respectively and were assigned to the respective treatments. The animal source
feed ingredients such as fish meal, squid meal, meat and bone meal, shrimp meal and fish oil and the plant origin feed ingredients like
wheat gluten, soybean meal, maize gluten and wheat flour were used for feed formulation. The feed additives such as vitamin and

Table 1
Feed formulation for development of feeds with varying levels of dietary protein.
Ingredients (g/kg) CP350 CP400 CP450 CP500

Wheat gluten1 0 20 20 30
Soybean meal2 150 240 101.5 50
Squid meal3 50 50 70 100
Meat and bone meal4 80 100 100 75
Maize gluten5 50 50 50 50
Shrimp meal6 70 80 100 70
Fish meal7 140 141 300 429
Wheat powder8 326 187 144 89.5
Fish oil9 56 56 45 40
Vitamin mix10,a 20 20 20 20
Mineral mix11,b 20 20 20 20
Sodium meta bisulphate12 1 1 1 1
Butylated hydroxyl toluene13 1 1 1 1
DL-Methionine14 4 4 2 0
Lysine15 7.5 5.5 1 0
Vitamin C16 2.5 2.5 2.5 2.5
Lecithin17 10 10 10 10
Ocimum sanctum leaf powder18 10 10 10 10
Choline chloride19 2 2 2 2

Composition of nutrients
Moisture (g/kg) 34.6 38.5 33.3 45.0
Crude protein (g/kg) 352.6 409.3 455.3 499.2
Crude fat (g/kg) 99.0 95.2 105.9 100.5
Total ash (g/kg) 131.6 150.1 161.9 168.5
DE (kJ/g) 15.86 15.65 15.61 15.59
DP/DE (mg/kJ) 20.89 24.64 27.47 30.26

Number of replicates n = 3 for each feed sample for proximate analysis.

8&18
From the local market, Kochi, India.
12&13
Hi-Media, Mumbai, India.
17&19
Hi-Media, Mumbai, India.
CP350, Crude protein 350 g/kg; CP400, Crude protein 400 g/kg; CP450, Crude protein 450 g/kg; CP500, Crude protein 500 g/kg; DE, Digestible
energy; DP/DE, Digestible protein /digestible energy.
1
Viveka Essence Mart, Wall Tax Road, Chennai, India.
2
SakthiSoya, Coimbatore, India.
3
King Fish Products Pvt. Ltd., Veraval, India.
4
Arogya Bio Proteins, Vellore, India.
5
Sukhjit Starch and Chemicals Ltd, Phagwara, India.
6
Khaja Mohideen Traders, Wall Tax Road, Chennai, India.
7
Raj Fishmeal and Oil Co., Malpe, Mangalore, India.
9
Crude sardine oil from Kiriyathan Trading Co., Narakkal, India.
10
Supplevite – M from Sarabhai Zydus Animal Health Pvt. Ltd, Vadodara, India.
11
Agrimin from Vibrac Healthcare India, Pvt. Ltd., Mumbai, India.
14
Evonik Degussa, Germany.
15
BestAmino, CJ Corporation, South Korea.
16
Stay – C from DSM Nutritional Technologies, Mumbai, India.
a
Composition of vitamin mix (quantity/kg): Vitamin A, 500,000 IU; Vitamin D3, 250,000 IU; Vitamin E, 2000 mg; Vitamin K, 1000 mg; Thiamin,
100 mg; Riboflavin, 2000 mg; Pyridoxine, 500 mg; Cyanacobalamin, 400 mg; Calcium Pantothenate, 2500 mg; Niacin, 4000 mg; Biotin, 4000 mg;
Folic acid 200 mg.
b
Composition of mineral mix (quantity/kg): Manganese oxide, 500 mg; Potassium iodide, 500 mg; Ferrous sulphate, 10 g; Zinc oxide, 1000 mg;
Copper sulphate, 250 mg; Cobalt carbonate, 2 mg; Sodium selenite, 10 mg; Chromium chloride, 100 mg; Calcium lactate, 250 g; Calcium phosphate
(monobasic), 350 g; Magnesium sulphate, 50 g.

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D.L. Prabu et al. Animal Feed Science and Technology 269 (2020) 114685

mineral mixture, antioxidant, mould inhibitor, choline chloride and Ocimum sanctum leaf powder as immunostimulant were incor­
porated in all the diets. The available levels of limiting amino acids such as methionine and lysine were determined in the diets and
supplemented as required in order to recompense their deficiency. As listed in the formulae (Table 1) feed ingredients were weighed
accurately and mixed thoroughly using a homogenizer (Hobart, HL200, USA). The homogenized mixture was added with required
quantity of water and extruded through laboratory scale twin screw extruder (Basic Technology Private Ltd., Kolkata, India) using 8
mm diameter die. The extrudates were dried in hot air oven (LABLINE SPW, India) at 50 ◦ C for overnight. Then cooled to room
temperature and crushed using mixer grinder (Preethi Blue leaf™, Chennai, India) in pulse mode. The crushed feed was passed through
2 and 3 mm sieve to collect 2 and 3 mm crumbles. This slow sinking crumble feed was stored in air tight container and fed to the
experimental animals.

2.4. Fish rearing

The fish were fed to apparent satiation twice daily (10:00 and 16:00 h) during the 13-week feeding trial. The amount of feed
consumed by the fish in each tank was recorded daily. Round the clock filtered water circulation was made through indigenous re-
circulatory filter assembly (Prabu et al., 2017) with 5 g/kg saline water and the flow-rate of water in each tank was kept at 5
L/min. The faecal matter and unconsumed feed were siphoned out daily and exchanged water was replenished with fresh 5 g/kg saline
water. The temperature of culture water was maintained at 27 ◦ C using 2 numbers of 1000 W thermostat (AquaZonic, China). Dis­
solved oxygen (DO) value was maintained approximately 5–5.5 mg/L, the ammonia content was about 0.2− 0.25 mg/L, nitrite was
0.1− 0.15 mg/L, nitrate level was 0.5− 0.6 mg/L and pH ranged from 8.0 to 8.2 during the experimental period.

2.5. Feed intake and growth calculation

The body weight of experimental fish was measured at intervals of 30 days to assess the growth. The fish were starved overnight
before taking the body weight. Fish were weighed at a precision of 0.1 g using electronic balance (Hercules HT SS V4, India). The
growth performance and feed intake of snubnose pompano was assessed using the following formulae:

Biomass gain (BG; in g) = Mean final weight (Wf) –Mean initial weight (Wi)

Weight gain percentage (WG%; in g %) = (BG/Wi) × 100

Specific Growth Rate (SGR) % = (LogeWf –LogeWi/t)× 100

Average daily growth (ADG; in g/day) = (Wf–Wi)/Experimental period (t)

Geometric mean body weight (WG, in g) = √(Wi×Wf)

Mean metabolic bodyweight (MBWG; in kg0.8) = (WG/1000)0.8.

Growthrate on metabolic body weight (GRMBW; in g/kg0.8/ day) = (Wf -Wi)/(MBWG×t).

Daily growth coefficient (DGC, in %/ day) = 100×(W1/3 1/3

f -Wi )/t.

FI as fed (FI asfed in g/fish/ day) = Total feed fed/ (n × t)

Absolute feed intake (FIABS; g DM/fish/ day) = FIasfed ×Dry matter (DM)/100

Feed intake of fish on % of body weight (FIPCT; %/day) = (FIABS/WG)×100

Feed intake per metabolic body weight (FIMBW; g DM/kg0.8/day) = FIABS/MBWG

Feed gain ratio on DM basis (FGR) = FIMBW/GRMBW

Feed conversion ratio (FCR) = FI as fed/ BG

Protein efficiency ratio (PER) =WG/protein fed

Protein productive value (PPV; in %) = [Retained protein (g)/ Protein fed (g)] × 100

Survival rate (%) = (Total number of fish harvested/Total number of fish stocked)× 100

2.6. Sampling and homogenate preparation

At the end of the feeding trial, all fishes were starved for 24 h, and then final body weight in each tank was weighed accurately. Two
fish from each replicate were anesthetized with clove oil at 50 mg/L water. After that the fishes were euthanized and liver, brain, gill
and intestine tissues were removed carefully. The intestinal contents were removed before weighing. It was homogenized with 0.25 M

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D.L. Prabu et al. Animal Feed Science and Technology 269 (2020) 114685

chilled sucrose solution in a plastic tube using mechanical tissue homogenizer (T18™ model, IKA® India Private Limited, Bengaluru,
India). The tube containing tissue sample was continuously kept in ice to avoid heating. The homogenate was centrifuged at 5000 rpm
for 10 min at 4 ◦ C in a refrigerated centrifuge (Remi, CM 12 PLUS, India). The supernatant was stored at − 80 ◦ C till the use. A 10 %
homogenate was prepared for the tissues of interest for the assay of digestive and metabolic enzymes. From all treatments two more
fishes were sacrificed for the collection of blood and serum from the caudal vein as described by Prabu et al. (2016) and subsequently
used for estimation of whole body composition. The fishes were sliced into pieces and oven dried at 105 ◦ C until there was no change in
the dry weight.

2.7. Body indices

Two fish from each tank were sacrificed and dissected out immediately for the determination of hepatosomatic index, viscer­
osomatic index, intra-peritoneal fat ratio, meat yield-head-on, meat yield-headless and fillet yield as given below:

Hepatosomatic index (HSI; g/100 g) = [Liver weight (g)/ Weight of fish (g)] × 100

Viscerosomatic index (VSI; g/100 g) = [Weight of viscera (g)/ Weight of fish (g)] × 100

Gastrosomatic index (GSI; g/100 g) = [Weight of stomach (g)/ Weight of fish (g)] × 100

Intra-peritoneal fat ratio (IPFR; g/100 g) = [Intra-peritoneal fat weight (g)/ Fish weight (g)] × 100

Meat yield-head on (MY-HO; g/100 g) = [Muscle weight with head (g)/ Fish weight (g)] × 100

Meat yield-headless (MY-HL; g/100 g) = [Muscle weight without head (g)/ Fish weight (g)] × 100

Fillet yield (FY; g/100 g) = Fillet weight (g)/ Fish weight (g)

Cephalic index (CI, g/100 g)= [Weight of the head (g)/ Fish weight (g)] × 100

2.8. Proximate composition

Another three fishes from each replicate of all the treatments were used for proximate analysis. The crude protein (Kjeldahl ni­
trogen × 6.25) was determined by the Kjeldahl method after acid digestion using a Kjeldahl System (FOSS Kjeltec 2300), total fat
/ether extraction was done using a Soxhlet System (FOSS Soxtec 2043), ash content was determined by incinerating the samples in
muffle furnace at 600 ◦ C for 4 h (AOAC, 2000a; method 942.05) and moisture content was estimated using hot air oven (AOAC, 2000b;
method 934.01). Similarly the proximate composition of experimental feeds was done. The DE content and DP content of the
experimental feeds were estimated by the methods of Atwater and Woods (1896) and in vitro pepsin digestibility method (AOAC,
2005; method 971.09) respectively.

2.9. Amino acid composition

The amino acid profile of feed and whole body of fish samples in triplicates were performed by reverse-phase high-performance
liquid chromatography (HPLC) (Waters, USA). Samples were hydrolyzed with 6 M HCl at 110 ◦ C for 24 h. Hydrolysates were added to a
C-18 column and amino acids were separated via reverse phase HPLC. Amino acids were quantified with a photodiode array detector
following post-column derivatization with ninhydrin (Smith, 1997).

2.10. Digestive and metabolic enzymology

The α-amylase activity on carbohydrate was estimated using dinitro-salicylic acid method (Rick and Stegbauer, 1974). Protease
activity was determined by the casein digestion method (Drapeau, 1976). Lipase activity was determined by the p-nitrophenyl
palmitate (pNPP) hydrolysis method derived from Katsivela et al. (1995). The aspartate aminotransferase (AST) activity was assayed in
different tissue homogenates as described by Wootton (1964). The procedure adopted for alanine aminotransferase (ALT) activity was
same as for AST activity except the substrate 0.2 M DL- alanine used instead of aspartic acid of ALT procedure.

2.11. Serum biochemistry

The total proteins, triglycerides, cholesterol and glucose in the serum samples were analysed using commercial diagnostic kits
(Tulip diagnostics Ltd, Goa, India) according to the manufactures procedure using spectrophotometer (MERCK-Thermo Electron,
Madison, WI, USA).

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D.L. Prabu et al. Animal Feed Science and Technology 269 (2020) 114685

2.12. Statistical analysis

Glass aquarium with indigenous re-circulatory assembly was the experimental unit for all treatments. For serological, enzymo­
logical and biochemical examinations, the mean values of 3 fish samples per aquarium were considered for analysis of variance. The
data were statistically analyzed by statistical package, SPSS version 16 (Carver and Nash, 2008). Comparisons among all the treat­
ments were done by one way Analysis of Variance (ANOVA). Comparison between the various parameters of all the treatments was
made using Duncan’s Multiple Range Test (DMRT) (Duncan, 1955). Comparisons were made at the 5 % probability levels and all
statements of statistical significance were based on P < 0.05 unless otherwise stated. The regression analysis (Zeitoun et al., 1976) was
performed to determine the break point in growth parameters, which represented the optimum dietary protein requirement for
snubnose pompano.

3. Results

3.1. Proximate composition and amino acid profile of feed

The proximate composition of experimental feed is given in Table 1. All the four experimental diets matched with its expected
protein composition. The diets CP350 and CP500 were slightly lower than its said level of fat content (100 g/kg). The amino acid
profile of experimental feed (on dry matter basis) is given in Table 2. The essential amino acids composition was slightly higher than
the non essential amino acid composition in all the experimental diets. The limiting amino acids such as lysine and methionine levels
were similar as it was balanced with feed grade amino acids in CP350-CP450 diets and it was slightly higher in CP500 due to higher
inclusion of fishmeal in this diet.

3.2. Growth performance and feed intake

The growth performance and feed utilization of fish fed with different levels of crude protein after 13-week of feeding trial is
showed in Table 3. There was a significant difference in all the growth parameters from different treatment groups (P < 0.05). Higher
weight gain (%) and SGR (%) were noticed in CP400 group and the least values were observed in CP350 group. In case of geometric
mean body weight (WG) and mean metabolic bodyweight (MBWG) CP450 showed higher response and the least response was noticed
in T1(35) group. Growth rate on metabolic body weight (GRMBW), daily growth coefficient (DGC) and average daily growth (ADG)
were higher in CP400 group and lower in CP350 group. The survival percentage of the fishes fed with varying level of dietary protein
was shown in Table 3.
There was significant difference in all the feed intake parameters of fish from different treatment groups (P < 0.05). The feed intake
as absolute feed intake (FIABS), FI on % of body weight (FIPCT) and feed intake per metabolic body weight (FIMBW) were low in CP500
group and high in CP400 group. In case of FCR, poor value of 2.20:1 was noticed in CP350 group and best FCR (1.73:1) was observed in
CP450 group which was not significantly different from rest of the treatment groups. Better PER was witnessed in CP400 group and

Table 2
Amino acid profile of experimental feeds (g/kg).
Amino acid composition (g/kg) CP350 CP400 CP450 CP500

Non-essential amino acid (NEAA) composition

Asparagine 27.1 29.3 31.0 32.4
Glutamine 54.4 57.8 61.8 64.9
Serine 10.2 12.0 11.7 12.6
Glycine 19.8 21.6 23.3 24.4
Alanine 10.5 10.7 12.2 15.9
Proline 42.9 44.5 48.7 52.0
NEAA 164.9 175.9 188.7 202.2

Essential amino acid (EAA) composition

Histidine 15.9 19.9 21.5 23.1
Arginine 16.9 21.8 22.3 23.8
Threonine 12.4 15.8 16.4 17.6
Tyrosine 14.7 17.5 18.5 19.6
Valine 15.5 16.2 19.3 19.9
Methionine 11.4 11.5 11.6 12.2
Cystine 6.9 7.8 8.4 8.9
Isoleucine 12.4 16.8 18.2 19.8
Leucine 23.4 26.5 29.8 30.4
Phenylalanine 15.5 18.9 21.4 25.2
Lysine 21.4 21.5 21.6 22.5
EAA 166.4 194.2 209.6 223.0

CP350, Crude protein 350 g/kg; CP400, Crude protein 400 g/kg; CP450, Crude protein 450 g/kg; CP500, Crude protein 500 g/kg.
Number of replicate n = 3.

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Table 3
Growth parameters of different treatment groups.
Parameter CP350 CP400 CP450 CP500 SEM Significance level

Growth
Initial weight (g) 17.20 ab 16.89 a 17.39 ab 17.99b 0.162 0.073
Final weight (g) 72.93a 91.58b 91.11b 83.30 ab 2.785 0.025
Weight Gain percentage (in g%) 324.07a 442.24c 423.65bc 363.15ab 16.866 0.007
SGR (%) 1.60a 1.87c 1.84bc 1.70ab 0.037 0.015
WG (g) 35.42 a 39.28b 39.80b 38.71b 0.651 0.040
MBWG (kg0.8) 0.028 a 0.031b 0.032b 0.031b 0.001 0.040
GRMBW (g/kg0.8/day) 21.84a 26.31c 25.71bc 23.43ab 0.627 0.010
DGC (%/ day) 20.64a 27.66b 27.30b 24.19 b 1.033 0.022
ADG (g/day) 0.62a 0.83b 0.82b 0.73ab 0.030 0.022

Feed Intake
Feed intake (g) 122.49ab 131.90c 127.74bc 115.00a 2.181 0.008
FI as fed (g/fish/day) 1.36 ab 1.46c 1.42 bc 1.27a 0.024 0.007
FIABS (g DM/ fish/day) 1.31b 1.41c 1.37 bc 1.22a 0.024 0.005
FIPCT (%/ day) 3.71b 3.59b 3.44b 3.15a 0.071 0.005
FIMBW (g DM/kg0.8/ day) 46.37c 44.91bc 43.12b 39.40a 0.890 0.005
FGR 2.12b 1.72a 1.68a 1.68a 0.065 0.008
FCR 2.20b 1.78a 1.73 a 1.76a 0.047 0.010
PER 1.30 ab 1.41b 1.28ab 1.14 a 0.040 0.086
PPV 26.07ab 28.18b 25.57ab 22.75a 0.788 0.082
Survival (%) 85.71 95.23 90.47 85.71 2.563 0.561

Glass aquarium with indigenous re-circulatory assembly was the experimental unit for all treatments.
Mean values in the same row with different superscript differ significantly (P<0.05).
Number of replicate n = 3; SEM, Standard error of the mean.
DGC, Daily growth coefficient; ADG, Average daily growth; FCR, Feed conversion ratio; FGR, Feed gain ratio on DM basis; FIABS, Absolute feed intake;
FIMBW, Feed intake per metabolic body weight; FIPCT, Feed intake of fish on % of body weight; GRMBW, Growth rate on metabolic body weight; PER,
Protein efficiency ratio; PPV, Protein productive value; SGR, Specific growth rate; WG, Geometric mean body weight; WG%, Weight gain percentage.
CP350, Crude protein 350 g/kg; CP400, Crude protein 400 g/kg; CP450, Crude protein 450 g/kg; CP500, Crude protein 500 g/kg.

least efficiency was recorded in CP500 group. No significant differences were observed on survival rate of different treatment groups (P
> 0.05) and better survival was recorded in CP400 group.

3.3. Body indices

There was significant difference on the various body indices of experimental fishes from different treatments (P < 0.05) except the
cephalic index (Table 4). The VSI, HSI, GSI and IPFR showed a decreasing trend as the crude protein content of the experimental diet
increases and the maximum value was witnessed in CP350 treatment. Higher percentage of meat yield on head-on as well as headless
condition was noticed in CP400 group followed by CP450 group and lower yield was noticed in CP350 and CP500 respectively. Higher
fillet yield was observed in CP400 group and lower yield was witnessed in CP350 group. The cephalic index was higher in CP450 group
and lower in CP500 group.

Table 4
Body indices of different treatment groups fed with graded levels of dietary protein.
Parameter CP350 CP400 CP450 CP500 SEM Significance level
c b b a
VSI (g/100 g) 5.33 5.16 5.19 4.85 0.055 0.001
HSI (g/100 g) 0.99 c 0.96b 0.95b 0.93 a 0.007 0.001
GSI (g/100 g) 0.40 c 0.37 b 0.36 b 0.34 a 0.006 0.001
IPFR (g/100 g) 1.32b 1.38c 1.34b 1.21a 0.027 0.001
MY-HO (g/100 g) 76.24a 79.01 b 78.90b 75.69a 0.505 0.003
MY-HL (g/100 g) 64.00a 66.88c 66.37 bc 65.05 ab 0.400 0.012
FY (g/100 g) 48.62a 51.08 b 50.89b 48.68a 0.405 0.009
CI (g/100 g) 12.25 12.12 12.53 10.65 0.367 0.288

Glass aquarium with indigenous re-circulatory assembly was the experimental unit for all treatments.
Mean values in the same row with different superscript differ significantly (P<0.05).
Number of replicate n = 3; SEM, Standard error of the mean.
HSI, Hepatosomatic index; VSI, Viscerosomatic index; GSI, Gastrosomatic index; IPFR, Intra-peritoneal fat ratio; MY-HL, Meat yield-headless; MY-HO,
Meat yield head-on; FY, Fillet yield; CI, Cephalic index.
CP350, Crude protein 350 g/kg; CP400, Crude protein 400 g/kg; CP450, Crude protein 450 g/kg; CP500, Crude protein 500 g/kg.

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D.L. Prabu et al. Animal Feed Science and Technology 269 (2020) 114685

3.4. Proximate composition and amino acid profile of pompano

The whole body proximate composition of pompano is given in the Table 5. There was a significant difference in whole body
moisture, crude fat and NFE content of different treatment groups (P < 0.05). Highest moisture content was noticed in CP350 group
and the lowest was observed in CP400 group. In case of crude fat, higher level was observed in CP400 and lower level was noticed in
CP350 group. There was no significant difference in crude protein and ash content of different treatments (P > 0.05). The crude protein
content also follows the identical trend as like crude fat content. The whole body amino acid profile of experimental fish is given in the
Table 6. There was significant difference (P < 0.05) in the serine, methionine, lysine and phenylalanine composition of whole body
content of snubnose pompano from different treatments and rest of the amino acids did not show any significant difference across the
treatments. The essential amino acid composition of whole body was slightly higher than the non essential composition in all the
treatment groups. The essential amino acid composition of whole body samples showed increasing trend as the protein content of
experimental diet increasing where as for non essential amino acids the reverse trend was noticed.

3.5. Digestive enzyme activity

The digestive enzyme activities of different experimental groups are given in Table 7. There was a significant difference in intestinal
and hepatic amylase and protease activity (P < 0.05). The α-amylase and protease activities were more in intestine than the liver of
different treatment groups. The amylase activity was higher in CP350 group and lower in CP500 group both in intestine and liver. The
maximum intestinal protease activity was noticed in CP400 group followed by CP450, CP500 and the least activity was in CP350
group. While comparing the protease activity of liver tissues with intestinal protease activity of different experimental groups, the
former was very meager and follows the similar trend as of later. There was no significant difference in lipase activity of both intestinal
and liver tissues of different groups (P > 0.05). The highest activity was found in CP350 group and lowest activity noticed in CP500
group in both tissues.

3.6. Metabolic enzyme activity

There was significant difference in AST and ALT enzyme activities of different treatment groups fed with graded level of protein (P
< 0.05) (Table 8). The activity of AST was higher in CP500 group and lower in CP350 group. The ALT activity was showed anin­
creasing trend as the protein level of diet increases similar to AST activity.

3.7. Serum biochemistry

Serum triglycerides, cholesterol and protein level were significantly differ (P > 0.05) among the different torments (Table 8). The
serum triglycerides and cholesterol level were inversely proportional to the dietary protein level in the diet. In case of serum protein as
the dietary protein increases the serum protein level was increased. There was no significant difference (P > 0.05) in blood glucose
level of snubnose pompano fed with graded level of dietary protein (Table 8). The glucose level was higher in lower protein fed groups
and the level got reduced as the protein content of the diet increased.

3.8. Optimize the protein requirement

The growth parameter, specific growth rate and feed utilization parameter, Protein efficiency ratio were subjected to second degree
polynomial regression analysis to determine the optimum protein requirement in snubnose pompano. Subjecting the data of specific
growth rate and dietary protein level to second-degree polynomial regression analysis, a break-point was evident at 426.7 g/kg of dry
diet, which was represented by the mathematical expression;

y = -0.004x2 + 0.349x - 5.664, R2 = 0.957

The relationship between protein efficiency ratio and dietary protein levels can be described by the second degree polynomial

Table 5
Whole body proximate composition of different groups of experimental fishes (as is basis).
Treatment CP350 CP400 CP450 CP500 SEM Significance level

Moisture (g/kg) 685.8b 671.5a 674.6a 673.8a 2.115 0.044

Crude Protein (g/kg) 195.6 203.5 202.4 200.4 1.359 0.165
Crude fat (g/kg) 67.3a 76.8b 67.6a 72.6ab 0.147 0.037
Total Ash (g/kg) 47.2 45.2 46.9 48.3 0.894 0.746
NFE (g/kg) 4.0a 2.9a 8.4b 4.8a 0.730 0.013

Mean values in the same column with different superscript differ significantly (P<0.05).
Number of replicate n = 3; SEM, Standard error of the mean.
NFE, Nitrogen free extract, CP350, Crude protein 350 g/kg; CP400, Crude protein 400 g/kg; CP450, Crude protein 450 g/kg; CP500, Crude protein
500 g/kg.

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D.L. Prabu et al. Animal Feed Science and Technology 269 (2020) 114685

Table 6
Amino acid profile of experimental fishes (on DM basis; g/kg).
Amino acids (g/kg) CP350 CP400 CP450 CP500 SEM Significance level

Non-Essential amino acid (NEAA) composition

Asparagine 65.1 64.4 64.3 64.8 0.172 0.322
Glutamine 107.3 106.2 106.1 106.6 0.189 0.073
Serine 28.1b 27.5b 26.4a 26.0a 0.289 0.003
Glycine 38.2 38.0 38.5 37.5 0.145 0.071
Alanine 62.0 61.5 61.1 60.3 0.253 0.082
Proline 31.7 31.1 30.4 29.8 0.269 0.148
NEAA 332.4 328.7 326.8 325.0 1.045 0.087

Essential amino acid (EAA) composition

Histidine 20.3 a 20.4 ab 20.1a 21.0b 0.128 0.044
Arginine 46.0 47.1 47.2 47.4 0.212 0.052
Threonine 22.6 23.3 22.9 23.8 0.214 0.775
Tyrosine 23.0 24.0 24.2 24.6 0.231 0.058
Valine 34.0 34.3 34.5 34.2 0.126 0.630
Methionine 21.0a 21.9b 22.0 b 24.2c 0.378 0.001
Cystine 4.3a 5.5b 5.8bc 6.0c 0.237 0.022
Isoleucine 33.0 33.3 34.2 34.0 0.202 0.091
Leucine 58.0 58.2 58.4 59.1 0.179 0.163
Phenylalanine 23.0a 24.3b 24.2b 24.4b 0.193 0.005
Lysine 61.9 a 63.4b 63.8b 65.2c 0.385 0.001
EAA 347.0a 355.7b 357.3 b 363.9c 1.099 0.010

Mean values in the same row with different superscript differ significantly (P<0.05).
Number of replicate n = 3; SEM, Standard error of the mean; DM, Dry matter.

Table 7
Digestive enzyme activities of experimental fishes fed with varying levels of dietary protein.
Digestive enzymes Tissue CP350 CP400 CP450 CP500 SEM Significance level
d c b a
Intestine 7.69 6.62 5.68 4.27 0.390 0.001
α-Amylase (nmol of maltose released/ min/mg protein)
Liver 3.89d 3.17c 2.51b 1.90a 0.228 0.001
Intestine 83.93a 101.01b 99.45 b 94.74 b 2.407 0.018
Protease (nmol of tyrosine released/ min/mg protein)
Liver 2.92a 5.46 b 5.51b 3.28ab 0.457 0.041
Intestine 4. 91 4.71 4.51 4.07 0.172 0.391
Lipase (unit/min/mg protein)
Liver 2.21 2.11 2.12 1.71 0.093 0.241

Mean values in the same row with different superscript differ significantly (P<0.05).
Number of replicate n = 3; SEM, Standard error of the mean.

Table 8
Serum biochemistry of experimental fishes fed with varying levels of dietary protein.
Parameters CP350 CP400 CP450 CP500 SEM Significance level
b b b a
AST (nmol of oxaloacetate formed/ min/mg protein) 0.54 0.53 0.51 0.45 0.013 0.038
ALT (nmol of pyruvate formed/ min/mg protein) 0.86 0.86 0.78 0.73 0.022 0.070
Glucose (mg/dL) 175.5 169.7 168.4 165.5 1.716 0.218
Triglycerides(mg/dL) 155.45c 143.28b 142.15ab 135.33a 2.399 0.002
Cholesterol (mg/dL) 107.44b 100.52b 102.17b 89.25a 2.213 0.007
Protein (mg/dL) 3.73a 4.62b 4.76b 4.89b 0.181 0.003

Mean values in the same row with different superscript differ significantly (P<0.05).
Number of replicate n = 3; SEM, Standard error of the mean.
AST, Aspartate aminotransferase; ALT, Alanine aminotransferase.

equation as follows; with a break point at 395.3 g/kg of dry diet.

y = -0.002x2 + 0.205x - 2.742, R2 = 0.927

The results of second degree polynomial regression analysis of SGR (%) and PER of different treatments (Figs. 1 & 2 ) revealed that
the crude protein requirement of snubnose pompano was in the range of 395.3–426.7 g/kg which was adequate to boost the growth of
the fish.

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D.L. Prabu et al. Animal Feed Science and Technology 269 (2020) 114685

Fig. 1. Second degree polynomial regression analysis of SGR in snubnose pompano fed with varying levels of dietary protein.
Number of replicate n=3
SGR, Specific growth rate

Fig. 2. Second degree polynomial regression analysis of PER in snubnose pompano fed with varying levels of dietary protein.
Number of replicate n=3
PER, Protein efficiency ratio

4. Discussion

Snubnose pompano is one of the fast growing candidate species for mariculture with good market demand. The culture of snubnose
pompano is being practiced in different locations with varying environmental conditions such as marine cages, coastal ponds and
inland low saline soil areas. But, the culture practice is still stumbling due to lack of species specific feed with optimum level of macro
nutrients. Among the nutrients, dietary protein is the key factor that affects the growth performance of fish and feed cost as well (Luo
et al., 2004). Protein comprises 600–700 g/kg of the dry matter in fish tissues, thus protein is the more indispensable nutrient in fish
nutrition (Wilson, 2002).
Jauncey (1982) found diminished growth rate at protein levels beyond the optimum requirement may be attributed that the fish
cannot effectively utilize the dietary protein once reaching the optimum protein level. In the present study, as the DP/DE and crude
protein content of the diet increases, the weight gain (%) and SGR (%) were increased until 400 g/kg CP in the diet and after 400 g/kg
CP the weight gain and SGR showed declining trend. This is in agreement with Lazo et al. (1998) who revealed that protein
requirement of juvenile Florida pompano is at 450 g/kg and generally the protein requirement of most of the marine fishes is in the
range of 400− 450 g/kg in a well balanced amino acid composition of the diet. In the current study, higher dietary protein fed groups
(450− 500 g/kg) yielded comparatively less growth. This is due to the fact that excess dietary protein leads to extra energy costs,
increased nitrogenous excretions and also higher feed cost (Monentcham et al., 2009). As like present study, Taynor (2013) reported
that higher SGR (%) at optimum DP/DE level in the diet of Trachinotus carolinus.
Reducing the protein content of fish feeds is one strategy to increase the sustainability of aquaculture through reducing feed costs as
well as reducing the environmental impact (Wilson, 2002). Protein requirement of fish is the sum of amino acid demands for protein
deposition, biosynthesis of metabolic intermediates and catabolism of substrates for energy. Several researchers demonstrated that
supplementation of one or more essential amino acids in the diets of fish improved the growth performance and feed efficiencies and
allow for reductions in total dietary protein content (Cheng et al., 2003; Yamamoto et al., 2005; Gaylord and Barrows, 2009).
Balancing the limiting amino acids in the protein-reduced diets decreases nitrogen excretion and improves nitrogen utilization in
several fishes. In the preset study, the fishes fed with 400 g/kg crude protein balanced with lysine and methionine showed better

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D.L. Prabu et al. Animal Feed Science and Technology 269 (2020) 114685

growth performance in terms of SGR, weight gain percentage and feed utilization than the higher level of protein fed group of fishes.
This is in agreement with Gaylord and Barrows (2009) who fed the rainbow trout with multiple amino acid supplementations to reduce
dietary protein in a plant-based diet. Similarly, Yamamoto et al. (2005) recorded similar protein efficiency in a 350 g/kg crude protein
diet supplemented with essential amino acids as like 450 g/kg crude protein diet.
In the present study, the GRMBW and DGC showed similar trend as like SGR (%) and was higher at 400 g/kg CP diet with balanced
level of limiting amino acids and optimum DP/DE content. Similarly, Saravanan et al. (2012a) found that excess dietary protein reduce
the GRMBW and DGC in Nile tilapia, Oreochromis niloticusas comparable to the present study. Further, Saravanan et al. (2012b) observed
better GRMBW and DGC in rainbow trout fed with higher DP/DE than the lower DP/DE content diet. This indicates that optimum
dietary protein and energy level yields better growth. Similarly, Fournier et al. (2002) revealed that the DGC was increased as the
protein content of diets increased in four teleosts such as rainbow trout, turbot, European seabass and gilthead seabream.
Feed intake is the main factor that determines animal growth. In fishes voluntary feed intake depends on nutritional, environmental
and physiological factors (Fletcher, 1984). Fish have been reported to show hyperphagia and consume more DE to pay off for reduced
dietary protein (Helland and Grisdale-Helland, 1998). In most of the carnivorous fishes, dietary protein levels beyond optimum level
do not seems to down-regulate DE intake (Geurden et al., 2006). In the present study, feed intake decreased with increasing dietary
protein and DP/DE ratio. These results were in line with El-Abed et al. (2013) and Lupatsch et al. (2001) who found decreased feed
intake in European seabass fed to satiation when dietary DP/DE ratio and DE level increased respectively. Similarly, Nguyen et al.
(2017) reported that higher dietary protein and DP/DE content in the diet of permit, Trachinotus falcatus showed reduction in feed
intake. However, feed intake in minimum level of protein (350 g/kg CP) fed group found lower which was due to comparatively lower
body mass than the other groups. In real sense, FI in minimum level of protein (350 g/kg CP) fed group was higher as per FIMBW. Thus,
in the present study, FIMBW was shown decreasing trend as the protein content of the diet increased. These results found true with
Saravanan et al. (2012a) who revealed that Nile tilapia fed with higher protein content consumed less feed on metabolic body weight
than the lower dietary protein fed counterparts. Conversely, Amin et al. (2014) found higher feed intake in brook trout, Salvelinus
fontinalis as the protein content of the diet increased.
In the present study, the PER was higher in optimum level of protein (400 g/kg CP) and declined as the protein content of the diet
increased to maximum (500 g/kg CP). This is in accord with Saravanan et al. (2012a) who revealed that Nile tilapia fed with lower
dietary protein content had better PER than the protein rich diet fed fishes. This is probably due to higher amount of dietary protein is
catabolised as a primary energy source when protein rich diets were fed to the fish (Kim et al., 1991). On the contrary, in brook trout
Amin et al. (2014) found higher PER for higher dietary protein fed group. Also, Lazo et al. (1998) observed extremely higher PER only
at 450 g/kg CP fed Florida pompano than the rest of the lower levels of dietary protein which were similar. FGR find similar trend in the
present study as like PER and this holds true with Nile tilapia (Saravanan et al., 2012a) and rainbow trout (Saravanan et al., 2012b). In
the present study, the PPV value was negatively correlated with dietary protein content of snubnose pompano fingerlings. These
findings were in line with Amoah (2012) in Arctic Char and Amin et al. (2014) in brook trout.
Fish biometrics such as hepatosomatic index (HSI), viscerosomatic index (VSI) and intraperitoneal fat ratio (IPFR) indicates the
body condition of fish. The HSI was found to be inversely proportional to the dietary protein levels and directly proportional to the
dietary carbohydrate levels (Yang et al., 2002; Tok et al., 2017). In the current study, HSI and VSI decreased with increase in dietary
protein level as the reduction of lipid deposition in the viscera and liver has been related to increased metabolic activity associated with
the intake of additional dietary proteins beyond optimum level (Phumee et al., 2009) and also reduction in deposition of excess glucose
into glycogen through gluconeogenic pathway due to lower dietary carbohydrate level in the diet (Yang et al., 2003; Tok et al., 2017).
This is in agreement with Kim et al. (2016) in parrot fish, Oplegnathus fasciatus, Aliyu-Paiko et al. (2010) in Channa striatus and Phumee
et al. (2009) in Pangasianodon hypophthalmus. Also, Luo et al. (2004) found no significant differences in HSI in juvenile grouper
(Epinephelus coioides) fed with diet contains different level of dietary protein and isoenergetic contents reared in floating net cages. The
IPFR ratio was higher at optimum level of dietary protein (400 g/kg) and found lower at higher dietary protein content fed groups (500
g/kg). This is in agreement with Li et al. (2007) who reported similar results of higher IPFR ratio at 320 g/kg CP and lower at 360 g/kg
CP in the channel catfish × blue catfish F1 hybrid catfish and Li et al. (2001) in different strains of channel catfish. Generally, the
processing yield increase as the dietary protein content increase until optimum level after which the processing yield will decrease due
to higher metabolic burden. In this study, higher muscle and fillet yield was achieved at 400 g/kg CP fed groups and found lower at 500
g/kg CP fed groups. This is in accord with the results of Li et al. (2001) in USDA103 and Mississippi “Normal” strains of channel catfish
fed with graded level of dietary protein. Therefore, it is an ideal strategy of feeding the snubnose pompano with optimum dietary
protein diets (400 g/kg) to enhance the fish production and to improve the processing yield that will be beneficial for marine fish
processor. This is because as dietary protein increases, the dietary DP:DE ratio becomes increased.
Nutrient retention in different fish species increases with increased dietary nutrient levels in feed. In the present study, the whole
body crude protein and lipid contents were increased with the increasing dietary protein levels which are in accord with Kim et al.
(2016) for juvenile parrot fish. Similar results were also obtained by Bai et al. (1999) for juvenile yellow pufferfish fed with graded
level of dietary protein and Tok et al. (2017) for P. hypophthalmus fed with different level of dietary protein on alternative day. On the
contrary, Phumee et al. (2009) reported that whole body crude lipid content of iridescent catfish decreases as the crude protein content
of whole body increases.
As like the crude protein content of the whole body tissues the amino acid composition was also influenced by dietary amino acid
profile in the present study. The limiting amino acids lysine and methionine were balanced in 350, 400 and 450 g/kg crude protein fed
diets and the level of these amino acids were slightly higher in 500 g/kg CP fed groups due to higher level of inclusion of fishmeal. The
slight increase in the lysine and methionine content in the diet of CP500 group fishes reflected trivial increase of these limiting amino
acids in the whole body muscle. This is in agreement with Kalhoro et al. (2018) who fed the juvenile of Acanthopagrus schlegelii with

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D.L. Prabu et al. Animal Feed Science and Technology 269 (2020) 114685

same level of dietary lysine and methionine showed similar level of accretion of these limiting amino acids in the dorsal muscle.
Digestive enzymes play a vital role in the hydrolysis of ingested nutrients to convert them into respective simple nutrients that can
be absorbed by the fish (Furne et al., 2005). The nutrient composition of the diet affects the digestive and metabolic enzyme profile of
the fish (De Silva and Aderson, 1995). Alpha amylase is one of the important carbohydrases which hydrolyse glycosidic links between
sugar residues especially starch and breaks down into glucose molecules. As snubnose pompano can utilize the carbohydrates
moderately well, the activity of amylase positively correlated with carbohydrate level in the diet and the activity increased with the
raise in carbohydrate content of the diet (Le Moullac et al., 1994). This is in accord with the results of Tok et al. (2017) who found
similar response of amylase activity in P. hypopthalmus and Xavier et al. (2016) in Labeo rohita. The protease activity is directly
influenced by the level of dietary protein, and the enzyme activity may dwindle beyond the required level of protein for the fish (Le
Moullac et al., 1994). In the present study, protease activity of the experimental fish fed 400 g/kg CP showed better activity which is
better than the higher protein diet fed groups. This result clearly corroborates the higher PER of 400 g/kg CP fed groupsthan the higher
protein fed groups. Similar result was reported by Tok et al. (2017) and Debnath et al. (2007) in different fish species. In the current
study, the activity of intestinal lipase enzyme found maximum at lower protein fed groups (which has equal contribution of animal and
plant source oil) and showed declining trend as the protein content of the diet increased (which has higher level of animal source oil).
This is may be due to better utilization of dietary lipid as an energy source owing to availability of corresponding lower level of protein
which otherwise used for energy production if found excess. The source of lipid, balance of animal and plant source lipid content and
species to be fed may also influence the level of lipase activity. This inference is in line with the findings of Kalhoro et al. (2018) who
fed the juvenile of A. schlegelii with plant and animal source oil at varying levels and found higher lipase activity at equal level of
animal and plant source oil. However, Tok et al. (2017) in P. hypophthalmus and Debnath et al. (2007) in L. rohita did not found clear
trend in the lipase activity according to the level of dietary protein.
Generally, teleosts prefer to use amino acids as primary source of energy than lipid and carbohydrates through gluconeogenic
pathway (Demeal, 1978; De Silva and Aderson, 1995). The AST and ALT enzymes have both protein degradation and synthesis effects
according to the feeding regimen prevail in fish. In the present study, the AST and ALT activities showed increasing activity as the
protein content of the diet increased which may be due to increased transamination of dietary protein (Pieper and Pfeffer, 1980). This
is in agreement with results of Tok et al. (2017) in iridescent catfish and Yengkokpam et al. (2007) in catla. The reason behind this
process is due to high protein intake beyond optimum requirement may enhances gluconeogenesis and amino acid catabolism
(Sanchez-Muros et al., 1998; Perez-Jimenez et al., 2007).
According to Melo et al. (2006) increased dietary protein level enacts the rise of serum protein which could be due to the
enhancement of digested protein in the circulatory system. In the present study, serum protein level was elevated due to increase in
dietary protein content. This was in harmony with the findings of Abdel-Tawwab et al. (2010) in Nile tilapia and Khalil et al. (2016) in
gold fish. On the other hand, Abou-Daoud et al. (2014) reported an increase in plasma protein level in lower protein fed marbled
spinefoot, Siganus rivulatus. In the present study, the serum triglyceride and cholesterol levels were inversely proportional to the di­
etary protein content. This was in agreement with the previous reports by Chen et al. (2009) in Genetically Improved Farmed Tilapia
strain of Nile tilapia (Oreochromis niloticus) and Yang et al. (2003) in Spinibarbus hollandi fed with varying dietary protein level. Blood
glucose is primarily produced from glycogenolysis or gluconeogenesis in the liver and transported to circulatory system. In the present
study, blood glucose values decreased with increase in dietary protein. The higher blood glucose level in the lower dietary protein fed
groups indicated that the glucose content is primarily derived from glycogenolysis due to higher level of liver glycogen content. This
was accord with the results of Khalil et al. (2016) in gold fish and Cheng et al. (2006) in orange spotted grouper, Epinephelus coioides.
However, Abou-Daoud et al. (2014) did not find any significant difference in blood glucose level in S. rivulatus fed with varying dietary
protein level.

5. Conclusion

In conclusion, after carefully examining all parameters related to growth, feed utilization and biochemical composition, feed with
400 g/kg CP, 100 g/kg crude fat and with available lysine (21 g/kg) and methionine (10 g/kg) performed better than rest of the
experimental diets. The second degree polynomial regression analysis of specific growth rate and protein efficiency ratio revealed that
the dietary protein requirement of snubnose pompano was in the range of 395.3–426.7 g/kg for optimal growth and better feed
utilization which is comparatively lower than the rest of the species of pompano. Hence, the diet CP400 can be effectively utilized for
culture of snubnose pompano in low saline water. Snubnose pompano appears to be a promising species for tropical mariculture due to
its better growth rate, relatively lower dietary protein requirement and readiness towards any (plant or animal source) formulated
feeds with better feed conversion efficiency.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

CRediT authorship contribution statement

D Linga Prabu: Conceptualization, Investigation, Writing - original draft. Sanal Ebeneezar: Data curation, Supervision. S
Chandrasekar: Visualization, Methodology. C.S. Tejpal: Validation, Methodology. M. Kavitha: Formal analysis, Writing - review &
editing. P Sayooj: Software. P. Vijayagopal: Writing - review & editing.

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D.L. Prabu et al. Animal Feed Science and Technology 269 (2020) 114685

Declaration of Competing Interest

The authors report no declarations of interest.

Acknowledgement

The authors are thankful to Director, Indian Council of Agricultural Research-Central Marine Fisheries Research Institute, Kochi for
his constant support to carry out the research work with the in-house funding. The laboratory work done by Mr. S. Nandhakumar Rao
(late) and Mr. B. Vishnu is also thankfully acknowledged.

Appendix A. Supplementary data

Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.anifeedsci.
2020.114685.

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