An approach to optimizing dietary protein to growth and body composition in walking catfish, Clarias batrachus (Linneaeus, 1758)

Abstract

Clarias batrachus is a commercially important food fish. In the present study, effect of varying dietary protein levels was evaluated on the survival, growth parameters and proximate composition of C. batrachus. Diets comprising 25%, 30%, 35%, 40%, 45%, and 50% crude protein (CP) were supplied to fish in T1, T2, T3, T4, T5, and T6, respectively, at the rate of 5% of fish body weight for the entire 90 days, twice daily. Size of each stocked C. batrachus was recorded after 15 days. Results revealed 100% survival rate of C. batrachus in all treatments. Significantly highest (P<0.001) mean value of weight gain (g/fish), percent weight gain, daily growth rate, specific growth rate and protein efficiency ratio (PER) in C. batrachus were recorded, reared in T4 by feeding 40% CP in diet. The best FCR value (1.90±0.02) for C. batrachus was obtained in T4 by feeding 40%CP in diet. Mean value of water, ash, fat and protein contents (wet mass) were ranged 74.10–79.23%, 3.12–4.68%, 3.90–4.43% and 13.09–16.79% for C. batrachus in the studied treatment groups. Water content (%) was found significantly (P<0.05) higher in the body of C. batrachus for T1, T2, T3 and T6 than for T4 and T5. Ash was found significantly (P<0.05) higher in the fish reared in T4 and T5. Fat content in the wet body mass of C. batrachus was found significantly higher in T4 and T1. While, significant higher (P<0.05) values of mean protein content was noted in C. batrachus reared in T4 and T5. Body composition of C. batrachus was also categorically affected by body size, however, condition factor showed non-significant correlation in most of the relationships in the present study. Overall, results indicated that feeding appropriate diet (containing 40% CP) to the fish resulted good growth performance, lower FCR and higher protein content in the fish. Present study provides valuable knowledge of optimal dietary protein level in C. batrachus which will help in commercial success of aquaculture.

Full text

RESEARCH ARTICLE
An approach to optimizing dietary protein to
growth and body composition in walking
catfish, Clarias batrachus (Linneaeus, 1758)
Zara Naeem
1
, Amina Zuberi
1
, Muhammad Ali
2
, Ammar Danyal Naeem
3
,
Muhammad NaeemID
3
*
1Fisheries and Aquaculture Program, Department of Zoology, Faculty of Biological Sciences, Quaid-i-Azam
University, Islamabad, Pakistan, 2Vice Chancellor, Quaid-i-Azam University, Islamabad, Pakistan,
3Institute of Zoology, Bahauddin Zakariya University, Multan, Pakistan
*dr_naeembzu@yahoo.com
Abstract
Clarias batrachus is a commercially important food fish. In the present study, effect of vary-
ing dietary protein levels was evaluated on the survival, growth parameters and proximate
composition of C.batrachus. Diets comprising 25%, 30%, 35%, 40%, 45%, and 50% crude
protein (CP) were supplied to fish in T1, T2, T3, T4, T5, and T6, respectively, at the rate of
5% of fish body weight for the entire 90 days, twice daily. Size of each stocked C.batrachus
was recorded after 15 days. Results revealed 100% survival rate of C.batrachus in all treat-
ments. Significantly highest (P<0.001) mean value of weight gain (g/fish), percent weight
gain, daily growth rate, specific growth rate and protein efficiency ratio (PER) in C.batrachus
were recorded, reared in T4 by feeding 40% CP in diet. The best FCR value (1.90±0.02) for
C.batrachus was obtained in T4 by feeding 40%CP in diet. Mean value of water, ash, fat
and protein contents (wet mass) were ranged 74.10–79.23%, 3.12–4.68%, 3.90–4.43% and
13.09–16.79% for C.batrachus in the studied treatment groups. Water content (%) was
found significantly (P<0.05) higher in the body of C.batrachus for T1, T2, T3 and T6 than for
T4 and T5. Ash was found significantly (P<0.05) higher in the fish reared in T4 and T5. Fat
content in the wet body mass of C.batrachus was found significantly higher in T4 and T1.
While, significant higher (P<0.05) values of mean protein content was noted in C.batrachus
reared in T4 and T5. Body composition of C.batrachus was also categorically affected by
body size, however, condition factor showed non-significant correlation in most of the rela-
tionships in the present study. Overall, results indicated that feeding appropriate diet (con-
taining 40% CP) to the fish resulted good growth performance, lower FCR and higher
protein content in the fish. Present study provides valuable knowledge of optimal dietary
protein level in C.batrachus which will help in commercial success of aquaculture.
1. Introduction
Walking catfish, Clarias batrachus, is one of the most well-known catfish species and a com-
mon food fish due to its lack of intramuscular bones, distinctive flavour and excellent
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OPEN ACCESS
Citation: Naeem Z, Zuberi A, Ali M, Naeem AD,
Naeem M (2024) An approach to optimizing dietary
protein to growth and body composition in walking
catfish, Clarias batrachus (Linneaeus, 1758). PLoS
ONE 19(5): e0301712. https://doi.org/10.1371/
journal.pone.0301712
Editor: Amit Ranjan, Tamil Nadu Dr J Jayalalithaa
Fisheries University, INDIA
Received: January 10, 2024
Accepted: March 20, 2024
Published: May 3, 2024
Peer Review History: PLOS recognizes the
benefits of transparency in the peer review
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all of the content of peer review and author
responses alongside final, published articles. The
editorial history of this article is available here:
https://doi.org/10.1371/journal.pone.0301712
Copyright: ©2024 Naeem et al. 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.
Data Availability Statement: All relevant data are
within the paper.
Funding: The author(s) received no specific
funding for this work.

nutritional content. Moreover, this fish can be easily stored and transported alive to markets.
As a result, consumers are always eager to pay more for this fish [1]. This commercially impor-
tant fish species is widely used as an aquaculture and marketed as live, fresh, and frozen due to
its high economic value as food fish [2]. It can live in a variety of low-oxygen settings, includ-
ing swamps and marshes, and burrows within the mudflat throughout the summer [3]. Adap-
tations like terrestrial dispersal, aerial respiration, and high tolerance to hypoxia and ammonia
can therefore be studied using C.batrachus as the ideal model [4].
Fish is considered as a crucial constituent of supportable diets for the future [5]. Subse-
quently, fish production stagnates, upcoming demand will depend on aquacultural products
[6]. Growing aquaculture production will need an upsurge production of fish feed [7] and to
improve the capability of farmed fish to assimilate feed consumption into biomass which can
help to reduce feed use in the fisheries industry and thus will enhance its sustainability through
condensed expenditures and ecological effects [8].
An important issue in fisheries industry is feeding, particularly when it affects production
costs and the health and growth of fish [9]. Feed expenses mark up 40–50% of total production
costs of fish [10]. Furthermore, growth optimization in the fish farming system is imperative
to confirm success [11]. Fish growth at all stages in its life history is mainly controlled by dif-
ferent factors, including feeding rate, food intake, feeding frequency, food type, and the ability
for nutrient absorption [12]. The capability to alter ingested feed into body mass growth can
be observed by the feed conversion ratio (FCR). It can be enhanced by modification in feed
composition [13]. As, FCR is a commonly used measure of conversion which is commonly
used over the entire production life of a fish, and obviously, the amount of feed changes during
this period [14], however, assessing the FCR in different life stages, like juvenile and younger
stages, may be helpful for better management.
In fish feed, protein is the largest nutrient and considered the most costly source for quality
health and optimum growth of a fish [15], but it also influences growth performance and feeds
conversion ratio of fish [16–18]. Fish usually ingest protein to attain non-essential and essen-
tial amino acids, essential for enzymatic function, muscle formation, and to supply energy
[19]. Though, both excessive and inadequate protein in the feed not only affects the quality
and growth of fish but also influence expenditure on aquaculture and as well as water quality.
Therefore, optimal dietary protein level is imperative for best growth and to support good
health in fish culture system [20].
Proximate body composition helps to rank different fish species based on their nutritional
and functional benefits and to assess the energy value of the fishes. Thus, allows consumers,
feed formulators and researchers to select the fish according to their requirements for their
nutritional values and/or processing [21–24]. The significance of proximate composition has
been discovered in the study of fish bioenergetics and the effect of pollutants. It acts as a good
indicator of fish physiology [25]. Fish proximate body composition constituents (fat, protein,
water, organic content and ash) are influenced by diet, feed rate, sex, genetic strain, age, species
and also by changing body size and condition factor [26–28].
The objective of the present study was to assess the survival rate, growth performance, feed
conversion ratio and proximate composition of Clarias batrachus fed on varying dietary pro-
tein levels.
2. Materials and methods
2.1. Ethics statement
2.1.1. Institutional review board statement. It has been confirmed that the experimental
data collection complied with appropriate permissions from Ethical Review Committee,
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Competing interests: The authors have declared
that no competing interests exist.

Department of Zoology, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad,
Pakistan. The study did not involve humans.
2.2. Experimental design
Fish fry of Clarias batrachus comprising 0.5–1.38 g wet body weight (W) and 3.90–7.40 cm
total length (TL) were procured from the Tawakkal Fish Hatchery & Farm, Muzaffargarh,
Pakistan, and acclimatized on rice polish in glass aquaria for two weeks. Feeding trial was con-
ducted in eighteen glass aquaria (volume 50 L), each having working dimensions of 60 x 40 x
44 cm
3
. After acclimatization, a total of 180 fish fry of C.batrachus were randomly stocked
comprising ten fish in each aquarium (30 per treatment) and three replicates were followed for
each treatment.
Six experimental diets (Table 1) comprising 25, 30, 35, 40, 45, and 50% crude protein (CP)
were supplied to the fish in treatment-1 (T1), treatment-2 (T2), treatment-3 (T3), treatment-4
(T4), treatment-5 (T5) and treatment-6 (T6), respectively. Proximate composition of experi-
mental feeds was calculated following the studies of NRC [29], Preston [30] and NDDB [31].
Feed was given to the fish at the rate of 5% of fish body weight for the entire 90 days of the
experimental period, twice (0900 and 1800) in two equal meals, with 16 hours light and 8
hours dark cycle daily. Dissolved oxygen and pH of water in each aquarium were monitored
daily. The temperature of each aquarium was maintained at 24–26˚C during the study period.
Size (W and TL) of each stocked C.batrachus were recorded after 15 days to adjust the feeding
rates and to calculate different growth parameters i.e. length gain, percent length gain, mean
final weight, weight gain, daily growth rate, percent weight gain, feed conversion ratio (FCR),
Table 1. Ingredients (%) used for feed formulation and proximate composition of various diets.
Ingredients T1
(CP-25)
T2
(CP-30)
T3
(CP-35)
T4
(CP-40)
T5
(CP-45)
T6
(CP-50)
Canola Meal 5 5 5 5 5 2
Corn Gluten Meal 30% 10 9 5 5 3 0
Corn Gluten Meal 60% 10 15 15 15 5 3
Fishmeal 10 10 15 21 30 37
Rice Polish 25 15 10 4 3 0
Sarson Meal 5 2 5 5 3 5
Soybean Meal 10 15 25 31 40 44
Sunflower Meal 5 5 5 5 3 2
Wheat Bran 15 19 10 4 3 2
Dicalcium Phosphate 1 1 1 1 1 1
Vitamin Premixes 1 1 1 1 1 1
Soybean Oil 2 2 2 2 2 2
Carboxymethyl Cellulose (CMC) 1 1 1 1 1 1
Proximate Composition of the Diets (%)
Moisture 8.96 9.28 8.95 8.25 8.86 8.9
Dry Matter (DM) 91.04 90.72 91.05 91.75 91.14 91.1
Crude Protein (CP) 25.15 29.95 30.00 34.80 40.10 45.03
Crude Fat (CF) 8.11 6.91 7.20 7.50 7.82 8.02
Ash 8.16 7.83 7.63 7.86 7.90 7.91
Fiber 8.38 8.05 8.10 8.03 8.22 8.07
Nitogen Free Extract (NFE) 41.24 37.98 38.12 33.56 27.10 22.07
NFE = DM-(%CP+ %CF + %Ash + %Fiber)
https://doi.org/10.1371/journal.pone.0301712.t001
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specific growth rate (SGR%) and protein efficiency ratio (PER). At the end of the feeding trial,
the total number of fingerlings in each tank was counted to calculate survival rate.
Growth performance of C.batrachus fed varying levels of dietary protein was measured as a
function of the weight gain by calculating the following statistics:
Percent Length Gain (%LG) = (Avg. final length−Avg. initial length /initial length) ×100
Percent weight gain (%WG) = (Avg. final weight −Avg. initial weight/initial weight) ×100
Daily Growth Rate (DGR) = Avg. final body weight −Avg. initial body weight/growth
period (in days)
Feed conversion ratio (FCR) = Dry feed intake (g)/biomass gain (g)
Specific growth rate (SGR%) = 100×(Ln final weight −Ln initial weight) / growth period
Protein efficiency ratio (PER) = Weight gain (g)/protein intake (g)
At the end of the feeding trial, fish specimens were immersed and kept in solution of
MS222 (250mg/L) for 10 minutes to euthanize the fish. Proximate composition was analysed
by taking of whole body of the fish. In brief, specimens of C.batrachus were dried to a constant
weight at 80˚C in an oven (Incucell, MMM Medcenter Einrichtungen GmbH, MMM-Group)
to determine water content in the fish. Ash was determined by incineration using muffle fur-
nace (RJM-1.8-10A) at 550˚C for 12 hr. Fat was determined by extracting in a chloroform and
methanol solution (1:2). Protein contents in C.batrachus were assessed by difference from
mass of other constituents, following the approach adopted by Naeem and Salam [27].
2.3. Statistical analyses
The data were subjected to ANOVA followed by Duncan’s new multiple range test to study the
differences among treatments in SPSS version 23. Mean differences among treatment were
determined by Duncan’s multiple range test and considered significant at p<0.05. Correlation
and regression analyses (Y = a + bX) were also performed to study the effect of fish size on
proximate composition in C.batrachus. Correlation coefficients for regression analyses were
considered significant at p<0.05, p<0.01 and p<0.001.
3. Results
Growth performance of the walking catfish (Clarias batrachus) fed with different feed treat-
ments (25%, 30%, 35%, 40%, 45%, and 50% CP) is presented in Table 2. Survival rate (%) of C.
batrachus was found 100% in all the studied treatment groups. Fortnightly length gain (cm)
and fortnightly weight gain (g) of C.batrachus in different studied treatments is represented in
Figs 1and 2, respectively.
Length gain and percent length gain (%LG) of C.batrachus showed non-significant differ-
ences (P>0.05) among various studied treatment groups. However, dietary protein levels sig-
nificantly affected (P<0.05) mean final weight, weight gain, daily growth rate, percent weight
gain, FCR, SGR%, and PER of C.batrachus (Table 2).
Significantly highest (P<0.001) mean value (6.96±0.04) of weight gain (g/fish) in C.batra-
chus was recorded in the fishes that were reared in T4 by feeding 40%CP in the diet. Daily
growth rate of C.batrachus was significantly highest (P<0.05) in T4 (40%CP) with a mean
(±SE) value of 0.63±0.005. The highest (P<0.001) mean (±SE) value of percent weight gain
(80.84±0.28%) was also recorded in the fishes that were fed 40%CP in T4, while that was the
lowest (56.14±1.04%) in T1 (25%CP), as shown in Table 2.
The best FCR value of C.batrachus was obtained in T4 by feeding the fish 40% crude pro-
tein in diet, as it remained the significantly (P<0.001) lowest (1.90±0.02) among various stud-
ied treatments. While the highest FCR value was recorded as 5.24±0.11 in T1, in which C.
batrachus were supplied 25% dietary protein level. Specific growth rate (SGR%) of C.batrachus
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was found significant highest (P<0.05) in T4(40%CP) with mean value being 0.80±0.01, while
the lowest (0.40±0.01) in T1 by providing fish 25% crude protein in diet. Highest (P<0.05)
mean value of protein efficiency ratio (PER) for C.batrachus was also recorded as 1.32±0.01in
feeding treatment which was supplied with a diet containing 40% protein level (T4). The low-
est mean PER value (0.60±0.02) for C.batrachus was found in T6 in which fish were supplied a
diet containing 50% protein.
Mean value of water, ash, fat and protein contents (% wet mass) were ranged from 74.10
±0.31% - 79.23±0.52%, 3.12±0.07% - 4.68±0.05%, 3.90±0.06% - 4.43±0.05% and 13.09±0.58% -
Table 2. Descriptive statistics (Mean±SE) for various growth parameters of Clarias batrachus fed upon various dietary crude protein levels in different treatments.
Parameters T1
(CP-25)
T2
(CP-30)
T3
(CP-35)
T4
(CP-40)
T5
(CP-45)
T6
(CP-50)
p-value
Survival rate of fish (%) 100 100 100 100 100 100 —
Mean Initial Length (cm) (iTL) 5.29±0.04
ns
5.25±0.02
ns
5.28±0.02
ns
5.31±0.05
ns
5.38±0.08
ns
5.27±0.09
ns
.692
Mean Final Length (cm) (fTL) 6.71±0.08
ns
6.59±0.03
ns
6.72±0.08
ns
6.67±0.09
ns
6.70±0.10
ns
6.60±0.10
ns
.793
Length gain (cm) (LG) 1.42±0.06
ns
1.32±0.04
ns
1.44±0.07
ns
1.36±0.04
ns
1.33±0.02
ns
1.33±0.07
ns
.439
Percent Length Gain (%LG) 21.15±0.63
ns
19.96±0.57
ns
21.42±0.78
ns
20.37±0.30
ns
19.79±0.03
ns
20.16±0.93
ns
.384
Mean Initial Weight (g) (iW) 1.37±0.01
ns
1.30±0.01
ns
1.37±0.02
ns
1.33±0.01
ns
1.33±0.02
ns
1.31±0.03
ns
.093
Mean Final Weight (g) (fW) 3.13±0.04
c
3.26±0.03
c
5.41±0.01
b
6.96±0.04
a
5.01±0.01
b
4.18±0.05
c
<.001
Weight Gain (g/fish) (WG) 1.76±0.06
c
1.96±0.02
c
4.04±0.01
b
5.63±0.05
a
3.68±0.04
b
2.87±0.04
c
<.001
Percent Weight Gain (%WG) 56.14±1.04
c
60.18±0.22
c
74.63±0.35
b
80.84±0.28
a
73.40±0.54
b
71.97±0.47
b
<.001
Daily Growth Rate (g/day) (DGR) 0.19±0.004
c
0.22±0.002
c
0.45±0.002
b
0.63±0.005
a
0.41±0.004
b
0.32±0.004
bc
<.001
Feed Conversion Ratio (FCR) 5.24±0.11
a
4.78±0.012
a
2.70±0.02
b
1.90±0.02
c
2.64±0.04
b
3.33±0.09
b
<.001
Specific Growth Rate (SGR%) 0.40±0.01
c
0.44±0.002
c
0.66±0.01
b
0.80±0.01
a
0.64±0.01
b
0.55±0.01
bc
<.001
Protein Efficiency Ratio (PER) 0.76±0.02
bc
0.70±0.002
c
1.06±0.01
b
1.32±0.01
a
0.84±0.01
b
0.60±0.02
c
<.001
Mean values sharing the same superscript in a row are not significantly different (p>0.05),
ns
= not significant
T1 = 25%CP, T2 = 30%CP, T3 = 35%CP, T4 = 40%CP, T5 = 45%CP, T6 = 50%C
https://doi.org/10.1371/journal.pone.0301712.t002
Fig 1. Fortnightly mean length (cm) of Clarias batrachus in different studied treatments.
https://doi.org/10.1371/journal.pone.0301712.g001
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16.79±0.27% in the studied treatments (Table 3). Analysis of variance (ANOVA) revealed sig-
nificant difference in all body constituents (both in wet and dry masses) among the studied
treatments (T1-T6). Water content (%) was found significantly (p<0.05) higher in the body of
C.batrachus for T1, T2, T3 and T6 than for T4 and T5. Ash (wet mass) was found significantly
higher in the fish reared in T4 (4.68±0.05%) and T5 (4.53±0.06%), while the lowest in T6 (3.12
±0.07%). Fat content in the wet body mass of C.batrachus was found significantly higher in T4
and T1. Significant higher value of mean protein content in wet mass was noted in C.batra-
chus reared in T4 (CP-40) and T5 (CP-45).
Water content (%) in the body of C.batrachus showed highly significant negative correla-
tion (p<0.001) with ash content (% wet mass) reared in T1 by feeding 25% of crude protein,
and significant (p<0.01) in T2, T3 and T6; while non-significant correlation was observed in
T4 and T5. Fat content was found significantly correlated (p<0.001) with water (%) only in T1
and T2. However, protein content of (p<0.001) showed highly significant negative correlation
(p<0.001) in all the studied treatment (Table 4).
Fig 2. Fortnightly mean weight (g) of Clarias batrachus in different studied treatments.
https://doi.org/10.1371/journal.pone.0301712.g002
Table 3. Mean values (±SE) of various constituents in percentage (%) of wet and dry mass of Clarias batrachus reared in different treatments.
Constituents T1
(CP-25)
T2
(CP-30)
T3
(CP-35)
T4
(CP-40)
T5
(CP-45)
T6
(CP-50)
p-value
Water (%) 78.81±0.74
a
79.23±0.52
a
78.33±0.30
a
74.10±0.31
b
75.50±0.50
b
78.97±0.40
a
<.001
Ash Wet mass 3.68±0.14
b
3.72±0.10
b
3.41±0.09
c
4.68±0.05
a
4.53±0.06
a
3.12±0.07
d
<.001
Dry mass 17.50±0.43
b
18.03±0.45
ab
15.73±0.34
c
18.14±0.25
ab
18.69±0.43
a
14.90±0.31
c
<.001
Fat Wet mass 4.42±0.11
a
3.95±0.09
c
3.90±0.06
c
4.43±0.05
a
4.18±0.07
b
4.22±0.07
ab
.002
Dry mass 21.22±0.52
a
19.15±0.36
b
18.09±0.34
bc
17.13±0.21
c
17.28±0.51
c
20.28±0.53
ab
.001
Protein Wet mass 13.09±0.58
b
13.09±0.41
b
14.35±0.26
b
16.79±0.27
a
15.80±0.51
a
13.70±0.40
b
<.001
Dry mass 61.29±0.83
c
62.82±0.62
bc
66.18±0.55
a
64.73±0.38
ab
64.03±0.86
b
64.82±0.76
ab
<.001
Mean values sharing the same superscript in a row are not significantly different (p>0.05)
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Size (wet weight and total length) of the fish represented highly significant correlation
(p<0.001) with all the body constituents in all the studied treatment groups for the studied cat-
fish, C.batrachus (Tables 5and 6).Water showed negative allometric pattern for all the studied
treatments groups with an increase in body weight of C.batrachus. Ash contents showed posi-
tive allometry for all the studied treatments groups except for T3 (CP35) which represented
isometric pattern (b = 1.036). Slope (b value) represented positive allometry for fats in the
body of C.batrachus in T1, T2 and T4, negative allometry in T6, while isometry in T3 and T5.
Positive allometry was also found in all treatments except for T5 (CP45) which represented
isometry with an increase in body weight of the studied fish (Table 5). On the other hand, all
the body constituents showed negative allometry for all studied treatment groups with an
increase in total length of C.batrachus (Table 6).
Table 7 showed that condition factor remained significantly correlated with water only in
T2 (r = 0.427, p<0.01), T3 (r = 0.618, = p<0.001) and T4 (r = 0.540, p<0.01). Ash content
was found insignificantly correlated (p>0.05) in all treatments except for T2 which was found
negatively correlated (r = 0.608, p<0.001) with condition factor of C.batrachus. Fat was also
found significant (r = 0.367, p<0.05) with condition factor for only T4, in which the fish was
fed a diet containing 40% crude protein. Significant correlation was also found in T3
(r = 0.636, p<0.01) and T4 (r = 0.507, p<0.001).
4. Discussion
Evaluation of the optimum dietary protein level is one of the most critical factors for the suc-
cess of aquaculture operations. In the present work growth performance of Clarias batrachus
was compared between different feeding treatments with six varying levels of formulated diets
Table 4. Statistical regression parameters of percentage water (%W) content versus % body constituents in wet mass of C.batrachus reared in different treatments.
Equation Treatment r a b SE of b t-Stat
%Ash = a+b%Water T1 -0.754*** 14.631 -0.139 0.023 -5.965
T2 -0.563** 12.657 -0.113 0.032 -3.538
T3 -0.552** 15.843 -0.159 0.046 -3.439
T4 -0.324
ns
8.729 -0.055 0.031 -1.783
T5 -0.144
ns
5.839 -0.017 0.023 -0.758
T6 -0.416** 8.506 -0.068 0.029 -2.382
%Fat = a+b% Water T1 -0.703*** 12.654 -0.105 0.020 -5.143
T2 -0.702*** 13.442 -0.120 0.023 -5.123
T3 -0.266
ns
8.139 -0.054 0.038 -1.434
T4 -0.324
ns
8.729 -0.055 0.031 -1.783
T5 0.081
ns
3.293 0.012 0.028 0.424
T6 0.105
ns
2.702 0.019 0.035 0.549
% Protein = a+b% Water T1 -0.967*** 72.715 -0.757 0.039 -19.600
T2 -0.965*** 73.900 -0.767 0.040 -18.993
T3 -0.909*** 76.017 -0.787 0.069 -11.345
T4 -0.968*** 80.655 -0.862 0.043 -20.081
T5 -0.982*** 90.869 -0.994 0.037 -26.654
T6 -0.959*** 88.793 -0.951 0.054 -17.500
r = Correlation Coefficient; a = Intercept; b = Slope; S.E = Standard Error
*** =p<0.001
** =p<0.01;
ns
>0.05
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containing 25% (T1), 30% (T2), 35% (T3), 40% (T4), 45% (T5) and 50% crude protein (T6).
Results of the growth experiment showed that the survival rate was recorded as 100% in all of
the studied groups, indicating high tolerance of the fish in the confined system and also repre-
sents that rearing conditions were good (optimal). Results of the present study agreed well with
a previously conducted study by Farhat and Khan [32], which reported a survival rate of 100%
in C.gariepinus by feeding the fish with 30%, 35%, 40%, 45%, and 50% crude protein (CP).
Results of present study also revealed that highest daily growth rate (0.63±0.005 g/day) was
observed in fish that were supplied 40% CP (T4) followed by 35% CP (0.45±0.002 g/day) in
T3, and the lowest (0.19±0.004 g/day) was found in fish fed on 25% CP in T1 for C.batrachus.
The observed difference in daily growth rate among the treatments was found to be statistically
(p<0.05) significant. Treatment groups showed difference in growth rate indicating the
importance of supplementary feeds on the growth and production of fish. In general, the daily
growth rate of C.batrachus recorded in the present experiment was found similar to that previ-
ously described by Tadesse [33], who have reported a daily growth rate of 0.23 to 0.52 g/day in
African catfish, Clarias gariepinus, fingerlings which were reared in tanks; but lower than that
of documented (1.12 g/day to 1.64 g/day) by Yalew [34], for different stocking density in C.
gariepinus. This lower growth rate of the fish than the study of Yalew [34] might be due to the
difference in feed composition of the ingredients or difference in fish size.
Table 5. Statistical regression parameters of log transformed wet body weight (g) versus log transformed total body constituents in wet mass of C.batrachus reared
in different treatments.
Equation Treatment r a b S. E. (b) tvalue when b = 1
Water = a+bWet Weight T1 0.983*** 0.019 0.746 0.027 -36.23
T2 0.984*** -0.005 0.811 0.028 -34.54
T3 0.992*** -0.018 0.879 0.022 -45.01
T4 -0.968*** 80.655 -0.862 0.043 -20.081
T5 0.957*** 0.079 0.712 0.042 -23.28
T6 0.971*** -0.017 0.863 0.041 -23.65
Ash = a+bWet Weight T1 0.960*** -1.906 1.956 0.109 -7.18
T2 0.830*** -1.637 1.399 0.181 -4.13
T3 0.680*** -1.498 1.036 0.215 -3.62
T4 0.798*** -1.450 1.143 0.166 -4.87
T5 0.841*** -1.432 1.124 0.139 -6.05
T6 0.845*** -1.798 1.465 0.178 -4.14
Fat = a+bWet Weight T1 0.963*** -1.661 1.624 0.088 -9.80
T2 0.927*** -1.676 1.530 0.119 -6.87
T3 0.817*** -1.400 0.987 0.134 -6.47
T4 0.905*** -1.849 1.587 0.143 -5.38
T5 0.718*** -1.369 0.983 0.183 -4.48
T6 0.676*** -1.233 0.769 0.161 -5.43
Protein = a+bWet Weight T1 0.916*** -1.410 2.062 0.173 -3.70
T2 0.921*** -1.304 1.818 0.148 -4.93
T3 0.959*** -1.313 1.641 0.093 -9.09
T4 0.877*** -1.482 1.838 0.194 -3.33
T5 0.718*** -1.369 0.983 0.183 -4.48
T6 0.820*** -1.325 1.736 0.233 -2.56
r = Correlation Coefficient; a = Intercept; b = Slope; S.E = Standard Error
*** =p<0.001
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FCR (Feed conversion ratio) is a vital gauge of the fineness of fish diet. A lower FCR desig-
nates better consumption of feed by a fish [35]. In the present research, the FCR values ranged
from 1.90 to 5.24 and varied significantly (p<0.05) between feeding treatments. Ogunji et al.
[36] declared FCR values of 1.2–1.5 as good range for fish raised with balanced diet and also
suggested that inclusion of more animal ingredients in fish diet may provide higher growth
rate and lower FCR of fish in the future. Although FCR value between 1.2 and 1.5 represent a
good indicator, it could not be used as absolute standard in all fish species, as it can change
according to several culturing factors. As, Tadesse [33] reported the lowest feed conversion
ratio (FCR = 2.14) in fish fed with 40% CP, indicating its suitability for African catfish finger-
lings than the other test diets. Further, in the present investigation, the lowest FCR value (1.90
±0.02) was noted in fish provided with 40% CP, representing that the fish consumed the feed
better than the other test feeds (25%, 30%, 35%, 45%, 50% CP) of the experiment for C.batra-
chus. The best FCR for C.batrachus in T4 might be due to the high proportion of protein
derived from easily digestible animal ingredients. FCR of the present study in C.batrachus is
very similar to those reported by Tadesse [33]. On the other hand, Ogunji and Awoke [37],
have reported lower FCR values of 1.61 for C.gariepinus reared in tanks under greenhouse.
Unlike these results of FCR in catfishes, Iqbal and Naeem [18], and Ishtiaq and Naeem [16],
noted the lowest food conversion ratio (FCR) fed upon 25% CP in the carp hybrid fry (Labeo
Table 6. Statistical regression parameters of log transformed total length (cm) versus log transformed total body constituents (g) in wet mass of C.batrachus reared
in different treatments.
Equation Treatment r a b S. E. (b) tvalue when b = 1
Water = a+bTotal Length T1 0.664*** -0.412 0.961 0.208 -13.48
T2 0.801*** -0.450 1.048 0.151 -18.84
T3 0.802*** -0.029 0.792 0.113 -25.65
T4 0.794*** 0.320 0.476 0.070 -42.32
T5 0.854*** 0.054 0.633 0.074 -39.87
T6 0.836*** -0.146 0.814 0.103 -28.43
Ash = a+bTotal Length T1 0.662*** -3.078 2.568 0.559 -2.80
T2 0.864*** -2.817 2.313 0.259 -9.25
T3 0.675*** -1.687 1.147 0.241 -11.31
T4 0.686*** -1.096 0.739 0.151 -19.16
T5 0.749*** -1.469 0.997 0.170 -16.69
T6 0.782*** -2.101 1.486 0.228 -11.69
Fat = a+bTotal Length T1 0.660*** -2.623 2.119 0.464 -4.35
T2 0.780*** -2.571 2.046 0.316 -7.46
T3 0.704*** -1.462 0.949 0.184 -15.36
T4 0.686*** -1.096 0.739 0.151 -19.16
T5 0.631*** -1.391 0.859 0.203 -13.91
T6 0.586*** -1.352 0.730 0.194 -14.72
Protein = a+bTotal Length T1 0.588*** -2.492 2.521 0.666 -1.98
T2 0.767*** -2.347 2.406 0.387 -5.34
T3 0.845*** -1.444 1.611 0.196 -13.67
T4 0.748*** -0.905 1.179 0.201 -13.74
T5 0.606*** -1.415 1.583 0.399 -5.93
T6 0.697*** -1.566 1.615 0.320 -7.77
r = Correlation Coefficient; a = Intercept; b = Slope; S.E = Standard Error
*** =p<0.001
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rohita ♀and Catla catla ♂) and carp (Catla catla), respectively. While Khalid and Naeem [38],
reported lowest FCR in grass carp (Ctenopharyngodon idella) by feeding only 20% protein in
diet. The variation might be due to differences in dietary habits of carps and catfishes.
In the present investigation, the SGR value was highest for C.batrachus fed with 40% pro-
tein and lowest for 25% dietary protein in fish. SGR increases with increasing dietary protein
levels up to 40% in C.batrachus and above optimum protein level, SGR decreased. These
results agree with the outcomes of Mohanta et al. [39] and Gandotra et al. [40] who also
reported that SGR increased with increasing dietary protein levels.
Present observation further reveals that fish-fed diet having 40% protein displayed signifi-
cantly (P<0.05) higher PER than those supplied with other dietary protein levels. Though a
propensity of growing PER values from 0.60 to 1.32 with the increase of each protein level in
diet up to 40% was noted, and afterward a significant drop in PER values were recorded in
diets containing higher protein level. Similar trend was also documented by Ahmed and
Ahmad [17], in Oncorhynchus mykiss fingerlings reared in the Himalayan region of India.
In the present study, whole body proximate composition of C.batrachus was categorically
affected by dietary treatment. Similar results were recorded for Pagrus pagrus, [41], Totoaba
Table 7. Statistical regression parameters of condition factor (K) versus percentages (%) of body constituents (wet mass, g) for C.batrachus reared in different
treatments.
Equation Treatment r a b S. E. (b) tvalue when b = 1
Water = a+bCondition Factor T1 0.019
ns
78.436 0.364 3.600 0.101
T2 0.427** 72.967 5.417 2.204 2.457
T3 0.618*** 74.220 2.211 0.541 4.085
T4 0.540** 70.019 1.685 0.505 3.338
T5 0.303
ns
72.029 2.010 1.218 1.651
T6 0.351
ns
75.625 2.203 1.131 1.948
Ash = a+bCondition Factor T1 -0.068
ns
3.923 -0.235 3.593 -0.065
T2 -0.608*** 5.509 -1.546 1.935 -0.799
T3 -0.203
ns
3.798 -0.209 0.674 -0.311
T4 -0.151
ns
4.876 -0.079 0.593 -0.133
T5 -0.235
ns
4.853 -0.188 1.242 -0.151
T6 -0.400*3.741 -0.411 1.107 -0.371
Fat = a+bCondition Factor T1 -0.122
ns
4.768 -0.338 3.574 -0.095
T2 -0.317
ns
4.743 -0.685 2.313 -0.296
T3 -0.041
ns
3.960 -0.030 0.688 -0.043
T4 -0.367*4.907 -0.199 0.558 -0.356
T5 -0.016
ns
4.202 -0.015 1.277 -0.012
T6 0.200
ns
3.868 0.230 1.183 0.194
Protein = a+bCondition Factor T1 0.014
ns
12.874 0.209 3.601 0.058
T2 -0.316
ns
16.781 -3.186 2.313 -1.377
T3 -0.636*** 18.023 -1.971 0.531 -3.713
T4 -0.507** 20.198 -1.407 0.517 -2.721
T5 -0.269
ns
18.916 -1.808 1.231 -1.469
T6 -0.324
ns
16.766 -2.022 1.143 -1.769
r = Correlation Coefficient; a = Intercept; b = Slope; S.E = Standard Error
*** =p<0.001
** =p<0.01
*=p<0.05;
ns
p>0.05
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macdonaldi [42], Culter alburnus Basilewsky [43], Catla catla [44], and Genetically Improved
Farmed Tilapia [45]. It is also documented that proximate composition depends on the fish
species, fish size, dietary protein sources and environmental conditions [46]. However, this
finding is in contrast to those reported in other studies for Seriola dumerili [47] and Solea sene-
galensis [48] and Siniperca scherzeri [49].
Present study revealed that mean value of water and protein contents were ranged 74.10–
79.23% and 13.09–16.79% in the studied treatment in which C.batrachus were fed with differ-
ent levels of dietary protein. These values fit within the range of those reported in previous
study for other strictly carnivorous fish species of the same genus, Clarias gariepinus [50]. Sig-
nificantly lower water content and higher whole-body protein contents were found in C.batra-
chus fed with a diet containing 40% CP and 45% CP than those of the fish fed with 25%, 30%,
35% and 50% CP in diets. However, Kim et al. [51] have reported no significant effect of die-
tary protein levels of 20%, 30% and 40% on crude protein of the Juvenile Catfish, Silurusasotus.
On the other hand, Ishtiaq and Naeem [44] have observed that dietary crude protein levels def-
initely affect the water and protein contents of Catla catla. Hence, the results indicated that
farmer can achieve not only higher growth and low FCR but can attain higher protein and
lipid contents in the C.batrachus by feeding the fish with 40% crude protein, rather higher
crude protein (45%CP) in diet.
Ash contents of C.batrachus fed with the experimental diets was also significantly affected
by dietary protein levels, which is in accordance with Hien et al. [52] for Clarias microcephalus.
The body lipid content generally increased as the dietary protein level increased in the present
investigation as previously reported by Bai et al. [53] for yellow puffer. On the contrary, Kim
et al. [51] documented that as the protein content of whole body increases, whole-body lipid
content decreases. However, in the present study, though, lipid contents were significantly
affected by dietary protein levels, but no increasing or decreasing effect was observed with an
increase in dietary protein. These discrepancies may be attributed to the difference in experi-
mental condition mainly dietary protein levels or due to fish species variation, as feed, inten-
sive feeding or starvation, maturity stage [54,55], sex [56], condition factor [57], age and
seasonal variations [58] and body size [59] have been found to have a pronounced impact on
the proximate composition of different fish species.
Literature shows that body weight of a fish influences the various body constituents [16], and
fat and protein increase while ash and water decrease with the increasing total length [59].
Hence, regression analyses were also performed to observe the effect of fish size on proximate
composition of C.batrachus. Highly significant correlation (p<0.001) between fish size (weight
and length) and body constituents (water, ash, fat and protein) were noted in the present work
and found in agreement to those reported by Naeem and Ishtiaq [60] for Mystus bleekeri, Bano
et al. [61] for Labeo calbasu and Ishtiaq and Naeem [16] for Catla catla. Negative allometric pat-
tern for water contents indicated an increase in this content with lesser proportion. While pro-
tein constituents were found increasing with greater proportion (positive allometrric pattern)
with an increase in fish size when compared with b = 1 for body weight and b = 3 for total
length. The negative allometry for water and positive allometry for protein is evident in many
studies [16,43,60,61] and hence the findings of present study indorse the same trend.
Furthermore, body composition of a fish species can be assessed from water content by per-
forming regression analyses. It allows to predict other constituents of body (protein, fat and
ash), with the consistent lessening of costs when performing one, in spite of diverse analyses.
These opinions have furnished by [16,62], and are in agreement with the results of the present
study, especially for protein contents in the body of C.batrachus which showed negative corre-
lation (p<0.001) in all the studied treatments, which is found in in conformity with the find-
ings of those reported by Bano et al. [61].
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Some studies documented a noticeable impact of condition factor on the proximate compo-
sition, however, most of the studies have reported insignificant relationships between body
constituents and condition factor, as body weight of a fish is not always proportional to the
cube of its total length [63]. The findings of the present study are in general agreement with
those reported with Naeem and Ishtiaq [60] in wild Mystus bleekeri, Khalid and Naeem [64] in
farmed Ctenophyrngodon idella and Kousar et al. [45] in Genetically Improved Farmed
Tilapia.
As, higher cost of a fish feed containing higher crude protein is considered a limitations or
challenge in implementing the recommended 40% crude protein diet on a commercial scale,
but farming industry should not compromise as it influences positively on the growth perfor-
mance, FCR and quality of fish as food. Moreover, delving the further research is recom-
mended to explore the lasting effects of employing the optimal 40% crude protein diet on C.
batrachus, considering aspects related to reproduction and overall health.
5. Conclusion
This investigation specifies that dietary protein levels affect the growth, FCR and proximate
composition of Clarias batrachus. The best growth parameters and chemical composition
(containing highest level of protein, mineral and fat constituents) can be achieved by feeding
the catfish with a diet comprising 40% crude protein (CP) than other dietary protein levels
(25%, 30%, 35%, 45% or 50% CP), and consequently, it is suggested that addition of 40% pro-
tein in feed is ideal for growth and effective feed utilization of the walking catfish, C.batrachus.
It is also evident that despite the variations, the mean values of percentage protein in different
treatments of this study indicates that C.batrachus is a good source of protein to consumers.
Moreover, body size shows a pronounced impact on body composition of fish. Data produced
in the current research would be beneficial in evolving nutritionally balanced diets for the
semi-intensive and intensive culture of this catfish.
Author Contributions
Conceptualization: Amina Zuberi, Muhammad Ali.
Data curation: Zara Naeem, Amina Zuberi, Ammar Danyal Naeem, Muhammad Naeem.
Formal analysis: Zara Naeem, Muhammad Naeem.
Investigation: Zara Naeem.
Methodology: Zara Naeem, Ammar Danyal Naeem, Muhammad Naeem.
Project administration: Amina Zuberi.
Resources: Ammar Danyal Naeem.
Software: Zara Naeem.
Supervision: Amina Zuberi.
Validation: Zara Naeem.
Visualization: Zara Naeem, Muhammad Ali.
Writing – original draft: Zara Naeem.
Writing – review & editing: Zara Naeem, Amina Zuberi, Muhammad Ali, Muhammad
Naeem.
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