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American Journal of  
Life Science and Innovation (AJLSI)

Whole Body Antioxidant Status of  Silver Carp Fingerlings Fed Diet 
Containing Various Dietary Organic Acids
Kanwal Razzaq1, Tariq Dildar2, Mudssar Aslam1, Sana Arif3

Volume 2 Issue 1, Year 2023
ISSN: 2833-1397 (Online)

DOI: https://doi.org/10.54536/ajlsi.v2i1.1349
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Article Information ABSTRACT

Received: March 01, 2022
Accepted: March 15, 2022
Published: March 21, 2023

A 10-week feeding trial was conducted to evaluate the whole- body antioxidant status of  
silver carp fingerlings when fed with diets containing various organic acids. Five 
experimental diets were formulated, such as D1 containing no supplemented organic acids, 
while D2 contain (2%malic acid), D3 (2%citric acid), D4 (2% formic acid) and D5 (2% lactic 
acid). During the experiment, water quality parameters including temperature, pH and 
DO were controlled. Results showed that acidification of  diet significantly reduced the 
activity of  SOD, CAT and GPX throughout the body of  silver carp fingerlings. Moreover, 
among different organic acid groups, the maximum value was observed in citric acid while, 
minimum value was recorded in formic acid. Data on whole -body antioxidant enzymes 
were subjected to one-way analysis of  variance following Steel et al. (1996). Differences 
between among means were compared by Tukey`s Honestly Significant Difference Test 
and considered significant at (p<0.05) (Snedecor and Conhran, 1991).

Keywords
Aquaculture, Silver Carp, 
Antioxidant Status, Citric 
Acid, Formic Acid

INTRODUCTION
The global aquaculture industry currently produces 45% 
of  all seafood that is consumed all over the world. It has 
been estimated that it will be increased by 75% in the next 
20 years (FTU, 2007). In Egypt, aquaculture accounts for 
60% of  total fish production sources (GAFRD, 2007). 
Essential nutrients are not enough to fulfill the demand 
of  the high quality feeds so complementary feed additives 
are needed to improve efficiency of  feed utilization and 
survival rates of  fish.
Among them, short chain organic acids are mainly used 
as they have beneficial effects on feed preservation and 
utilization (Luckstadt, 2006; Atapattu and Senevirathne, 
2013; Sing et al., 2014). Organic acid supplementation 
in fish diet reduces the gastric pH (Baruahet al., 2005) 
which leads to increase the absorption of  nutrients 
(Boling- Frankenbachet al., 2001) and also help in the 
breakdown of  phytate (Jongbloed, 1987), so it is easily 
available to fish. Moreover, it also decreases the gastric 
emptying rate by dietary acidification (Mayer, 1994). 
Furthermore, it also improves the gut health of  animal as 
they have antagonistic effect on microbes (Ravindran and 
Kornegay, 1993; Partanen and Mroz, 1999).
Organic acid increases the availability of  dietary 
minerals through acidification in several ways. Firstly, 
by the modification of  mineral transport mechanism by 
changing the stomach acidity. Secondly, supplementation 
of  organic acid in the diet acts as a chelating agent 
and affects the complex forming ability of  elements 
(Ravindran and Kornegay, 1993). Thirdly, the absorption 
area for minerals is increased (Baruah et al., 2007a) through 
the proliferation of  the epithelial cells in gastrointestinal 
mucosa (Sakata et al., 1995) by the inclusion of  organic 

acid in the diet.
CA, one of  organic acid is widely used for acidification 
of  diet as it has unique flavor and high buffering capacity 
(Hossain et al., 2007). It has a great potential to reduce 
the antagonistic interactions between trace elements by 
their chelating effect and also enhances the absorption of  
other trace minerals (Sugiura et al., 1998).
Proteolytic enzymes are stimulated by CA which leads to 
increase the feed intake, reduces the activity of  microbes 
including ammonia and also has the capacity to reduce 
the risks of  subclinical infections (Chowdhury et al., 2009; 
Ou et al., 2013).

Objectives of  this study
The aim of  present study was to investigate the whole 
body antioxidant status of  silver carp fingerlings fed 
acidified diets

MATERIALS AND METHODS
Experiment was performed in Fish Nutrition Laboratory, 
Department of  Zoology, Wildlife and Fisheries, 
University of  Agriculture, Faisalabad.

Fish and experimental condition
Before onset the experiment, silver carp fingerlings 
were obtained from Government Fish Seed Hatchery, 
Faisalabad. For acclimatization to indoor conditions, 
fish were placed for two weeks in tiled tanks (1000 L). 
During this period basal diet was given for 6 days (Allan 
& Rowland, 1992) to fish. For feeding trial, 9 species 
of  fish with same initial weight (3.526±0.0056 g) were 
kept in V shaped tanks (70L). Throughout this time, 
fingerlings were fed of  that basic food once a time in a 

1 Department of  Zoology, Wildlife and Fisheries, University of  Agriculture, Faisalabad, Pakistan
2 Department of  Zoology Division, Institute of  Pure and Applied Biology, Bahauddin Zakariya University Multan, Pakistan
3 Department of  Animal Breeding and Genetics, University of  Agriculture, Faisalabad, Pakistan
* Corresponding author’s e-mail: hasilpur910@gmail.com

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day. For each test diet experiment repeated three times. 
Feeding trial sustained for two months. During the study 
period, Jenway pH meter 3510 and DO 970 were used 
to monitor changings in water quality, specially dissolved 
oxygen, temperature and pH. All water tanks are inflated 
by capillary system around the clock.

Feed Ingredients and Experimental Diets
Feed Ingredients and feed stuff  obtained from local 
poultry market and composition of  chemical analysis was 
done by using standard methods (AOAC, 1995). Before 
incorporating feeding trial, the constituents of  food were 
crushed and filtered to achieve the desired particle size 
(Table I).
The method for pretreatment of  ingredients, 1 kg of  
the ground constituents such as fish meal, wheat flour, 
sunflower meal, corn gluten meal and 1.5L of  distilled 
water was adding for made paste and retained it for 38 
°C for 16 h and then dried. All dry ingredients were 
mixed for 15 minutes in an electric mixture. Though 
continuously stirring, then gradually add mineral mixture, 
vitamin premix and fish oil.
Five trial diets were made by using various supplementing 
organic acids at level of  2%. The D1 contains no 
supplemented organic acids, while D2 contain malic 
acid, D3 citric acid, D4 formic acid and D5 lactic acid, 
correspondingly. To prepare appropriate dough for each 
trial feed, slowly mix 10% to 15% water. To make floating 
particles, then further process it via a laboratory extruder. 
After particles are dried, they are crushed and sieved to 
the desired size. Keep the pellets in the refrigerator at -18 
° C until the feeding test is completed.

Chemical analysis of  feed
With help of  pestle and mortar fish samples and diet were 
standardized Methods for determining moisture was: 
drying in an oven at 105°C for 12 hours, micro Kjeldahl 
apparatus used for measuring crude protein. By Soxhlet 
system, crude fat through petroleum ether extraction 
method determined (Bligh & Dyer, 1959) and in an electric 
furnace for 12 hours crude fiber measured (Table II)

Table 2: Chemical composition (%) of  experimental diet
Diet Organic acids DM 

(%)
CP 
(%)

CF 
(%)

Ash 
(%)

D1 Control 89.49 31.11 9.05 9.95
D2 Malic acid 90.1 30.51 9.1 9.62
D3 Citric acid 90.17 31.071 9.045 9.8
D4 Formic acid 89.63 31.12 8.94 9.78
D5 Lactic acid 90.11 30.84 9.16 9.86

Table 1: Formulation (%) of  feed ingredients
Ingredient Percentage
Fishmeal 25
Sunflower meal 20
Soybean meal 10
Corn gluten meal 15
Fish oil 7
Rice Polish 10
Wheat flour 9
Mineral mixture** 1
Vitamin premix* 1
Organic acid 2
Total 100

Feeding Procedure and Sample Collection
For experimental feeding trial, the fingerlings were fed of  
their suggested diet. After feeding time of  three hours, 
the extra food was exhausted by opening the tank valve. 
Wash water tank thoroughly to eliminate particles from 
the food then fill-up the water. Afterwards, return the fish 
to the fish tank. After the two-hour interval, feces were 
collected in beaker by opening valves of  tank. In an oven 
at 60oC, each of  the repeatedly processed feces was dried. 
Then ground and stored for chemical inquiry. For each 
repeated sample, the trial was continued to collect 5 g of  
feces.

Figure 1: Modified UA system showing steel tank and 
main pipe

Figure 2: Modified UA System showing valve I and valve 
II and collection tube

Determination of  antioxidant enzymes
Preparation of  enzyme extract
Whole body was taken and rinsed with phosphate buffer 
of  pH 6.5 (0.2 M) and homogenized in cold buffer (1:4 
w/v) using a blender. After the homogenization, organ 
homogenates were centrifuged for 15 minutes at 10,000 rpm 
and 4ºC. After centrifugation process, clear supernatants 
were stored at 80ºC for enzyme assay while residue was 
discarded.

Enzyme Assay of  Superoxide dismutase
The activity of  superoxide dismutase was determined 

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Am. J. Life Sci. Innov. 2(1) 33-37, 2023

by measuring its ability to inhibit the photo reduction of  
Nitrobluetetrazolium (NBT) following the method of  
Giannopolitis and Ries (1997).

Procedure
1 ml buffer was taken in cuvette as blank and inserted into 
spectrophotometer to note the readings of  blank, after 
taking reading spectrophotometer was adjusted at zero at 
A560 nm. Then 5-6 cuvettes were taken and set them in a 
light box with an internally mounted light bulb of  30 Watt. 
Firstly 1 ml of  buffer was added to each cuvette, then 0.05 
ml enzyme extract and 0.016 ml of  riboflavin was added 
in each cuvette. All the cuvettes were incubated in light 
box for 12 minutes. The cuvettes were transferred to the 
spectrophotometer, where 0.067 ml of  EDTA/NaCN 
solution and 0.033 ml of  NBT was added to the illuminated 
reaction mixture. The absorbance was noted after 20 s of  
reaction. Activity of  superoxide dismutase was determined 
by measuring the % age inhibition of  NBT.

Enzyme Assay of  Catalase and Glutathione Peroxidase
The activity of  peroxidase was determined by measuring 
its ability to reduce the concentration of  H2O2 at A470 
nm (Civello et al., 1995).

Procedure
A cuvette containing the 2 ml of  blank solution was 
inserted into the spectrophotometer and set it to zero at 
wavelength of  240 nm. Then a cuvette containing buffered 
substrate solution was put into the spectrophotometer 
and initiation of  reaction was occurred by adding 0.05 ml 
of  enzyme extract the initiation of  reaction was occurred. 
The reaction time is 3 minutes and noted the absorbance 
after interval of  1 minute.

Calculation

Statistical Analysis
Finally, data regarding the activities of  antioxidant 
enzymes of  whole body were subjected to one-way 
analysis of  variance following Steel et al. (1996). The 
differences among means were compared by Tukey`s 
Honestly Significant Difference Test and considered 
significant at (p<0.05) (Snedecor and Conhran, 1991). 
Costate Computer Software, Version 6.303 was used for 
statistical analysis.

RESULTS
Superoxide dismutase status (U/mg protein) in 
whole body of  silver carp fingerlings
Effect of  different organic acids on whole body 
superoxide dismutase status in silver carp fingerlings is 
shown in table III. Data showed that acidification of  

diet significantly decreased the activity of  SOD in whole 
body of  silver carp fingerlings. Furthermore, maximum 
value was observed in formic acid while, minimum value 
was recorded in malic acid among different organic acid 
groups.

Table 3: Effect of  supplementation of  different organic 
acids on superoxide dismutase status (U/mg protein) in 
whole body of  silver carp fingerlings
Diet Organic acid SOD
D1 Control 4.55a

D2 Malic acid 3.65c

D3 Citric acid 3.83bc

D4 Formic acid 4.10b

D5 Lactic acid 4.02b

PSE 0.068
P-value 0.0018**
Data are means of  three replicates, P<0.05 Organic acids
PSE = pooled SE = √MSE/n (where MSE= mean-squared error)

Catalase status (U/mg protein) in whole body of  
silver carp fingerlings
Effect of  different organic acids on whole body catalase 
status in silver carp fingerlings is shown in table IV.Data 
showed that acidification of  diet significantly decreased 
the activity of  CAT in whole body of  silver carp 
fingerlings. Furthermore, maximum value was observed 
in malic acid while, minimum value was recorded in lactic 
acid among different organic acid groups.

Table 4: Effect of  supplementation of  different organic 
acids on catalase status (U/mg protein) in whole body of  
silver carp fingerlings
Diet Organic acid CAT
D1 Control 65.66a

D2 Malic acid 58.52b

D3 Citric acid 53.22c

D4 Formic acid 50.72c

D5 Lactic acid 50.05c

PSE 1.427
P-value 0.0025**
Data are means of  three replicates, P<0.05 Organic acids
PSE = pooled SE = √MSE/n (where MSE= mean-squared error)

Glutathione peroxidase status (mU/mg protein) in 
whole body of  silver carp fingerlings
Effect of  different organic acids on whole body 
glutathione peroxidase status in silver carp fingerlings 
is shown in table V. Data showed that acidification of  
diet significantly decreased the activity of  GPX in whole 
body of  silver carp fingerlings. Furthermore, maximum 
value was observed in citric acid while, minimum value 
was recorded in formic acid among different organic acid 
groups.

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DISCUSSION
Currently, there is considerable interest in the commercial 
use of  organic acids in fish diets, both to control disease 
and to enhance growth performance. Researches have 
reported that several organic acids, their salts or mixtures 
thereof  can improve growth, feed utilization and disease 
resistance in fish (Baruah et al 2007; Hossain, Pandey & 
Satoh 2007; Sarker, Satoh & Kiron 2007; Luckstadt 2008).
Hormones, antibiotics, ionospheres and some salts are 
most commonly used as growth promoters to increase the 
productive performance of  fish (Fuller, 1992; Go´ngora, 
1998; Klaenhammer and Kullen, 1999). Antibiotic 
growth promoters are used in fish diets as feed additives 
to favor growth and environment friendly aquaculture. 
However, it is necessary to make use of  these antibiotic 
growth promoters in less amount as feed additive in fish 
diets worldwide to enhance the development of  cross 
resistances to humans. However, the supplementation of  
organic acid is beneficial to replace the feed additives.
(Kim et al. 2006) investigated that organic acid 
supplemented diet can improve the growth and 
performance of  fishes. Fish feed industry has been using 
organic acids for decades where most extensively used 
acidifiers contain lactic, citric, fumaric and formic acid.
(Baruah et al. 2007) designed a factorial experiment to 
study the effect of  dietary microbial phytase, citric acid 
and their interactions on growth performance of  Labeo 
rohita juveniles. Hence, it is suggested that phytase 
and citric acid act synergistically to improve growth 
performance and nutrient digestibility of  L. rohita 
juveniles.
(Hossain et al. 2007) conducted a feeding experiment to 
investigate the effects of  organic acids on the growth 
and phosphorus utilization in fish. Therefore, it can be 
concluded from these results that organic acids can play 
an important role in development of  highly required eco- 
friendly diets.
(Pandey & Satoh 2008) observed phosphorus (P) and 
nitrogen (N) retention in rainbow trout fed on low fish 
meal based diets supplemented with organic acid. Hence, 
results conclude that low fishmeal based diets may prove 
beneficial for growth of  rainbow trout if  supplemented 
with certain organic acids.

Phromkunthong et al. (2010) determined the combined 
effect of  citric acid and microbial phytase on phosphorus 
utilization. Cyprinus carpio fingerlings were treated with 
citric acid and phytase for 60 days. It can be concluded 
that positive effects of  phytase can be achieved by adding 
low dose of  citric acid.

CONCLUSION
Findings of  experiment are as follows:

• Supplementation of  organic acids significantly 
(p˂0.05) decreased the whole body superoxide dismutase 
activity (U/mg protein) in silver carp fingerlings.

• Supplementation of  organic acids significantly 
(p˂0.05) decreased the whole body catalase activity (U/
mg protein) in silver carp fingerlings.

• Supplementation of  organic acids significantly 
(p˂0.05) decreased the whole body glutathione peroxide 
activity (mU/mg protein) in silver carp fingerlings.
In conclusion, supplementation of  organic acids decreased 
the antioxidant enzymes in silver carp fingerlings.

Authors Contribution
K.R planed, did experiments and wrote manuscript. T.D, 
S.A, M.A and S.A helped in experimental work.

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Table 5: Effect of  supplementation of  different organic 
acids on glutathione peroxidase status (mU/mg protein) 
in whole body of  silver carp fingerlings
Diet Organic acid GPX
D1 Control 75.82a

D2 Malic acid 70.11b

D3 Citric acid 71.46b

D4 Formic acid 68.27b

D5 Lactic acid 71.39b

PSE 0.904
P-value 0.0149*
Data are means of  three replicates, P<0.05 Organic acids
PSE = pooled SE = √MSE/n (where MSE= mean-squared error)

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