Immune response and growth performance of African Catfish (Clarias Gariepinus

Burchell, 1822) fed dietary lemon (Citrus Limon) oil

Oladapo Segun Michael, Oyinola Azeezat Odunayo. Federal University of Technology,

Akure, Nigeria. +2348160619942, +2348130822339. oladapo765@gmail.com &
yettyoyinola@gmail.com

Abstract

Essential oils such as Lemon oil has growth promotion, appetite stimulation,
immunomodulatory and antioxidant effects as well as antiparasitic, antibacterial, anaesthetic
and antistress activities in catfish. This study investigated the efficacy of lemon oil fed African
catfish (Clarias gariepinus) on the growth performance, mineral composition, blood profile,
proximate composition and cholesterol level. The experimental fishes has an average of 3.51g
and were subjected to 5 treatments for 70 days with the following levels of lemon oil inclusion;
0.0%, 0.4%, 0.8%, 1.0% and 1.2%. Feed intake, water quality parameters and growth
performance were recorded weekly. The results obtained and recorded by the study indicates
that the addition of lemon oil in fish diets decreased significantly (P<0.05) the weight gain
(13.48±0.48), decreased the specific growth rate (2.46±0.06), increased the feed conversion
ratio (FCR) (1.61±0.06), decreased the protein efficiency ratio (0.62±0.02) and increased the
survival rate (84.44±4.44) from the control (13.91±2.14), (2.50±0.17), (1.56±0.07),
(0.64±0.03) and (73.33±6.67) respectively. For the mineral composition of the catfishes, there
was a normal distribution of the mineral constituents even when the lemon oil was
supplemented increasingly to the diet. The blood profile showed that lemon oil significantly
improved some of the blood components (PCV, Hb, WBC, RBC, MCHC, MCV and MCH) of
the experimental fish. Also, the cholesterol level was decreased significantly with the addition
of lemon oil. However, increased growth performance and a well boosted immune response
was observed in all the experimented catfishes, an indication of a greater chance of survival
and adaptation. ) on the growth performance and biochemical parameters of African Catfish
(Clarias gariepinus). In this study five (5) different inclusion levels of lemon oil in the fish
feed, ranging from 0 % to 1.2 % were used in feeding the fish for seventy (70) days. Growth
performance parameters, including mean weight gain, specific growth rate, feed conversion
ratio, and survival rate, were evaluated. Additionally, serum biochemical parameters such as
aspartate transaminase (AST), total protein (TP), catalase (CAT), superoxide dismutase (SOD),
and lipid peroxidation (LPO) were analyzed. The results showed that the inclusion levels of
lemon oil had a significant impact on the growth performance of African Catfish. However,
the mean values of most growth parameters, including mean weight, mean final weight, mean
weight gain, specific growth rate, feed intake, feed conversion ratio, and feed efficiency ratio,
did not differ significantly across the different inclusion levels of lemon oil. Only the survival
rate exhibited significant differences, with the 0.4 % inclusion level demonstrating the highest
survival rate among the treatments. Biochemical analysis revealed that the inclusion of lemon
oil at 0.8 % resulted in significantly higher values of AST, CAT, and SOD, indicating improved
liver function and antioxidant defense mechanisms in the fish. Conversely, the total protein
levels were significantly lower at the 0.4 % inclusion level, suggesting a potential decrease in
protein synthesis. The lipid peroxidation levels remained relatively low and did not vary
significantly among the treatments. Overall, the findings suggest that the optimal inclusion
level of lemon oil in the fish feed for African Catfish may be around 0.8 %, as this resulted in
improved growth performance, enhanced liver function, and increased antioxidant defense
mechanisms. These results indicate that the essential oil from Lemon can serve as a beneficial
dietary supplement for African Catfish in aquaculture practices. Further studies are
recommended to explore the mechanisms underlying the observed effects of lemon oil on fish
growth and biochemical parameters. Additionally, investigations into the long-term effects,
optimal dosage, and potential interactions with other dietary components would provide
valuable insights into the practical application of lemon oil in aquaculture.
KEYWORDS: Clarias gariepinus, Lemon oil, growth performance, immune response.

Introduction

Aquaculture is a fast-growing activity on all continents, at a rate of around 7% per year,

accounting for more than half of the fish used in human consumption (Engle, 2016; FAO, 2016;
Engle et al., 2017). Aquaculture is therefore an important source of food, nutrition, income and
livelihood for hundreds of millions of people around the world. In 2014, the world production
from aquaculture was 73.8 million tons, with an estimated value of more than US$ 160 billion.
Of this total, 49.8 million tons (67.5%) were fish, with a value of US$ 99.2 billion (FAO, 2016).
Freshwater fish farming contributes more than two-thirds of the world’s aquaculture
production (Bastos Gomes et al., 2017). Its growth has been influenced by the increase in world
demand for foods of protein origin and for white meat, as well as by the high market value of
different fish species. Moreover, freshwater fish farming is an excellent alternative for
minimizing the intense wild fish exploitation that occurs in diverse regions of the planet. In
addition, current growth of the global population has been leading to additional expansion of
intensive fish production, because of demand for food (Engle et al., 2017; Soler-Jiménez et al.,
2017). In fish farming, fish populations are undergoing continuous selection to improve the
economic efficiency of this animal production. However, a number of both external and
internal factors affect fish production, and these problems unequivocally include parasitosis
(Soler-Jiménez et al., 2017).
According to Azambuja et al., (2011), the main aim of aquaculture is to maintain fish health
as well as to improve fish performance. Antibiotics are commonly used to control fish disease,
enhance the immune systems and as a growth promoter (Yilmaz and Ergun, 2014). However,
the usage of antibiotics has been restricted in many countries due to the resistance inducted to
bacteria and the residues that they could release in aquatic products (Citarasu, 2010). Therefore,
researchers in aquatic life are focusing on alternatives to the use of antibiotics in aquaculture.
Essential Oils (EOs) have potential to become a new generation of products for animal nutrition
and health, replacing antibiotic growth promoters in animal diets due to their positive effects
on digestion, gut microbial community, growth performance and welfare (Brenes and Roura
2010; Bento et al. 2013; Zeng et al., 2015). Essential oils are a natural blend of organic
substances characterized by a strong fragrance, synthesized by aromatic plants during
secondary metabolism. They are limpid (rarely coloured liquids), soluble in lipids and organic
solvents (insoluble in water) and generally have lower density than water (Bakkali et al. 2008;
Carson and Hammer 2011).
These ingredients are commonly added to fishes consumed raw and after cooking, especially
in the Mediterranean basin (Goulas and Kontominas, 2007; Ozogul, Polat and Ozogul, 2004).
Aquaculture have been recognized as the important sources for protein, minerals and vitamins
in many developing countries (Munglue et al., 2019. Fish is a very important source of animal
protein in the diets of man and aquaculture sector has showed a rapid growth in the last 30
years (Acar et al., 2015). Nigerians are large consumers of fish and it remains one of the main
products consumed in terms of animal protein as it is cheap and highly acceptable with no
restrictions (Tao and Linchun, 2008). In addition, the consumer demand for fish products has
been dramatically increasing for decades, leading to an increase in overfishing and intensive
fish culture around the globe.
The African catfish, Clarias gariepinus, a member of the family Clariidae, is an important
cultivated fish species (Ng and Romano, 2013). The worldwide high demand and preference
for the freshwater fishes, Nile tilapia and African catfish, can be attributed to factors such as
their low prices, rapid growth, accepting easily the formulated rations, tolerance to bad
environmental conditions, and their resistance to diseases (Iheanacho et al., 2017). The African
catfish is most popular among the contemporary fish types in Nigeria and has been used for
several studies in the aquaculture sector of the nation's agricultural base. The study will greatly
address the aspect of using EOs as immune boosters for Clarias gariepinus.

Materials and methods

Study area and experimental design

The experiment was carried out at Fishery Teaching and Research Farm of the Department of
Fishery and Aquaculture Technology, Akure, Ondo State. The experimental design is a
complete randomized design. Fifteen (15) glass tanks were arranged in triplicate. Fifteen (15)
fingerlings of approximately the same size were sorted, weighed and randomly distributed into
each glass tank (70cm x 50cm x 50cm) containing 45 liters of water. The average initial weight
of the fish was taken using a digital weighing balance (Metler Toledo, PB800 London).

Experimental system and fish

Fifteen (15) glass tanks were arranged in triplicate. Fifteen (15) fingerlings of approximately
the same size were sorted, weighed and randomly distributed into each glass tank (70cm x
50cm x 50cm) containing 45 liters of water. The average initial weight of the fish obtained
from the hatchery of the Fisheries and Aquaculture Teaching and Research Farm, FUTA, was
taken using a digital weighing balance (Metler Toledo, PB800 London) to be 3.51 ± 0.03g.
Each treatment was in replicate groups of three. They were acclimated for seven (7) days while
being fed the basal diet. The juveniles were not fed for 24 hours before started on the
experimental diet to maintain a uniform stomach condition of the fish and to induce their
appetite for the commencement of the feeding trial. During the feeding trial, fish were fed to
satiation with their respective diets twice daily between 8:00 am and 4:00pm. Feed were
administered bit by bit to check the rate at which the fish picked the feeds. The weight of the
fish in each tank was taken and recorded every week. Their agility (activeness) was checked at
each feeding period and water changed regularly to ensure the water was conducive for growth
and survival of the fish.
The lemon oil used for the research was ordered and imported from El Hawag Ltd in
Egypt. Other ingredients such as fishmeal, groundnut cake, corn flour soya bean meal, yellow
maize, lycine, methionine and fish oil were purchased from Farm Support Service Limited,
Akure, Ondo State.

Feed Preparation
Five isonitrogenous (40% crude protein) diets were prepared containing fish meal, groundnut
cake, soya bean meal, yellow maize and corn flour. These ingredients were grounded and
blended into powdery form and measured into five different bowls. Then, warm water was
added and mixed evenly into different containers to make a dough. The dough was pelletized
using Hobart pelleting Machine (Hobart Model 200 CA, USA) to get uniform size pellets
(2mm) to form the basal diet. The pelleted feeds were dried for 3days. The basal diet without
lemon oil serves as the control. Lemon oil was added in graded levels ranging from 0.4%,
0.8%, 1.0% and 1.2% to form diets 2 to 5. All dietary formulated feeds were packed in labelled
polythene bags and stored at -20℃ until use.

Table 1: The Gross Ingredients Composition (g/100g) of the Experimental Diets

Compositions Diet 1
Diet 2 Diet 3 Diet 4 Diet 5
(control)

Fish meal (65%) 25.8 25.8 25.8 25.8 25.8

Soyabean meal (44%) 25.0 25.0 25.0 25.0 25.0

Yellow maize (10%) 16.6 16.2 15.8 15.6 15.4

Groundnut cake (45%) 25.0 25.0 25.0 25.0 25.0

Fish oil (6%) 6.0 6.0 6.0 6.0 6.0

Methionine (1%) 0.4 0.4 0.4 0.4 0.4

Lysine (1%) 0.2 0.2 0.2 0.2 0.2

Corn flour (4%) 1.0 1.0 1.0 1.0 1.0

Lemon oil (%) 0 0.4 0.8 1.0 1.2

Total (%) 100 100 100 100 100

Monitoring of Water Quality
Culture water was changed twice in a week at the early hour of the day (morning) to maintain good
water quality. Temperature was measured with thermometer (YSI-DO 550 U.S.A.), pH was
measured with a pH meter (Hanna H198106 model), and Dissolved oxygen was measured using
dissolved oxygen test kit (JPP-607 model).

Growth and Nutrient Utilization Indices

Calculation of the performance data was according to Takeuchi, (1988) and Tacon, (1990). At the
end of the experiment, fish were counted and weighed. The growth parameters and feed utilization
indices were calculated as follows
Weight Gain (g) = Final weight – Initial weight

Specific growth rate (SGR)

This was calculated from data on changes of body weight over a given time interval; SGW (%per
(log W2 −log W1) ×100
day) =
𝑇2−𝑇1

Feed intake
This was obtained by adding daily mean feed intake (DFI) of fish under each treatment for the
experiment period.

Feed Conversion Ratio (FCR) = Feed intake (g) / weight gain (g)

Feed Efficiency Ratio (FER) = Weight gain (g) / feed intake (g)

Survival Ratio (%) = Number of fish harvested / number of fish stocked × 100
Proximate Composition of Experimental Feed

Proximate analysis was carried out on the formulated feed and experimental fish according to
AOAC, 2010 to determine the Moisture, ash, crude protein, crude lipid, crude fibre and Nitrogen
Free Extract (NFE). Gross energy (GE) was calculated as 5.65, 9.45,4.1kcal/g for protein, fat and
carbohydrate respectively (NRC, 1993)

Determination of Moisture Content

Two grams were weighed into crucible of a known weight and were kept in the oven at 105oC for
24 hours until a constant weight was obtained. The weight of the crucible plus the dried sample
was subtracted from the initial weight of crucible and sample. The results were recorded as
percentage of the initial weight sample to obtain the percentage moisture as follows

Loss of weight during drying

% Moisture  x 100
Weight of sample before drying

Where (Loss of weight = Initial weight of fish sample – Final weight of fish sample)

Determination of Ash content

1g of finely ground sample was weighed into a crucible of a known weight and kept in muffle
furnace at 3500C for 24 hours until a whitish- grey ash was obtained. The crucible was then placed
in the desiccators to cool and later weighed. The weight of the ash sample was subtracted from the
initial weight of the sample and the percentage ash was obtained as follow:

W2  W3
% Ash  x 100
W2  W1

Where: W1 = Weight of crucible

W2 = Initial weight of sample
W3 = Final weight of ash sample

Determination of Crude Fibre

Crude fiber was determined as loss due to ignition of dried lipid free residue after digestion with
1.25% H2SO4 and 1.25% of NaOH.
𝑊𝑒𝑖𝑔ℎ𝑡𝑜𝑓𝑟𝑒𝑠𝑖𝑑𝑢𝑒−𝑊𝑒𝑖𝑔ℎ𝑡𝑜𝑓𝑎𝑠ℎ×100
%Crude Fibre = 𝑊𝑒𝑖𝑔ℎ𝑡𝑜𝑓𝑠𝑎𝑚𝑝𝑙𝑒
Determination of Crude Protein
The crude protein content was determined using the micro-kjeldahl distillation method as
described by AOAC (1990). The percentage crude protein was calculated by multiplying the total
nitrogen by a factor of 6.25

M x T x 0.014 x V 1 x 100
% N
W V2

Where: N = Nitrogen content

M = Molarity of acid,
T = Control titre,
W = Weight of sample,
V1 = Volume of digest 1,
V2 = Volume of digest 2 while 0.014 is constant.
% crude protein = N × 6.25

Determination of Lipid Content

This was obtained by using the soxhlet apparatus. Two grams of oven dried samples were taken
and the lipid content was extracted with 40oC to 60oC petroleum ether for 8hours on a heating
mantle to extract the samples. The ether extracts were evaporated and the residue was weighed.
The percentage lipid content was calculated thus

W4  W3
% Fat  x 100
W2

Where W4 = Weight of flask + Extract

W3 = Weight of flask only
W2 = Weight of sample only

Determination of Nitrogen Free Extract (NFE)

This is the measurement of carbohydrate available in the sample. It was calculated by subtraction
method
NFE (%) = 100 – P – W – F – A
Where: P = Percentage protein (nitrogen x 6.25)
 W = Percentage water (moisture)
F = Percentage fat
A = Percentage ash

Mineral Composition: Determination of Ca, Mg, P, Mn AND Zn

The procedure of AOAC (1990) was used. The digest of the ash of each sample above as obtained
in calcium and potassium determination was washed into 100ml volumetric flask with de-ionized
/distilled water and made up to mark. The diluents were aspirated into the (Buck 200 Atomic
Absorption Spectrophotometer (AAS) made in Germany) through the section tube. Each of the
trace mineral elements was read at their respective wavelengths with their respective hollow
cathode lamps using appropriate fuel and oxidant combination. Each element was calculated as
milligram per kilogram (any of the elements) as:
𝑚𝑔 .
𝐸𝑙𝑒𝑚𝑒𝑛𝑡 ( ) = 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑥 𝑆𝑙𝑜𝑝𝑒 𝑥 𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝐹𝑎𝑐𝑡𝑜𝑟 1000
𝑘𝑔 .

Hematological Parameter Analysis

After terminating the experiment, 24 hour after the last feeding, blood was collected from 3
randomly selected fish from each treatment tanks with a 5-ml sterile hypodermic syringe and
carefully transferred into sterile EDTA (ethylene di-amine tetra acetic acid) heparinized tubes and
a sample bottle at room temperature. The EDTA bottles with blood samples was taken to the FUTA
Health Center, for hematological analysis while the whole blood in the sample bottle was taken to
the Department of Biochemistry, FUTA for serum analysis.
Determination of haemoglobin (Hb) content, leucocyte count (WBC), erythrocyte count (RBC).
Hb was estimated by the Cyanmethemoglobin method. RBC and WBC were counted by the
method of Rusia and Sood (1992) using hemocytometer. Erythrocyte indices of fish are; MCV
(Mean cell volume), MCH (Mean cell hemoglobin) and MCHC (Mean cell hemoglobin
concentration) were also calculated according to standard formula described by Svobodova et al
(1991) and total cholesterol (mmol/L) (TCHO), plasma glucose, Superoxide dismutase (SOD) and
catalase (CAT) was determined according to the method described by Svobodova et al (1991).
Haemoglobin Determination
The Haemoglobin was determined using the Cyanmethemoglobin method, the fish blood was
mixed with 5ml of Drabkin`s reagent which contains ferricyanide and cyanide. The ferricyanide
oxides changes haemoglobin to methemoglobin. The methemoglobin united with the cyanide to
form cyanmethemoglobin. The cyanmethemoglobin produced a color which is measured in
colorimeter at a wavelength of 540-nm in a spectrometer.

White Blood Cell Determination (WBC)

Blood smears was made within 45 minutes of sample collection, stained with Wright- Giesma, and
used to determine the WBC+ thrombocytes count and differential WBC counts. For differential
count, WBC and thrombocytes was counted until 200 WBC are enumerated on blood smears, and
the percentages of each WBC type was multiplied by the total WBC + thrombocytes count to
obtain the absolute differential cell count percentage.

Red Blood Cell Determination

The glacial acetic acid lyses the red cells. The blood specimen was diluted 1:200 in R.B.C. pipette
using Toission’s solution as diluting fluid and cells was counted under high power (40 X objective)
using a counting chamber. The red blood cells in the four corner squares and in the center square
(marked in the diagrams as ‘R’) was counted as the number of red cells per cu mm (µl) of whole
blood.
The following erythrocyte related indices was calculated:

Mean corpuscular volume (MCV)

The mean corpuscular volume was determined by calculation and the value gotten was recorded
in µm3 /cell
𝑃𝐶𝑉
𝑀𝐶𝑉 = × 10
𝑅𝐵𝐶
Where PCV - Packed cell volume/Haematocrit
RBC- Red Blood Cell

Mean corpuscular hemoglobin (MCH)

The mean corpuscular haemoglobin was determined by calculation and the value gotten was
recorded in pg/cell
 𝐻𝐵
𝑀𝐶𝐻 = × 100
𝑅𝐵𝐶
Where HB - Haemoglobin
RBC- Red Blood Cell

Mean corpuscular hemoglobin concentration (MCHC)

The mean corpuscular haemoglobin concentration was determined by calculation and the value
gotten was recorded in g l-1
𝐻𝐵
𝑀𝐶𝐻𝐶 = × 10
𝑃𝐶𝑉
Where PCV - Packed cell volume
HB- Haemoglobin

Serum Biochemical Analysis

Determination of Cholesterol
For the determination of cholesterol blood samples were collected in duplicate form each
treatment, the blood was centrifuged to exact the serum for about 15 minutes / 1000R evolution
per minutes using international equipment company IEC clinical centrifuge model 03645 SE
(IITA) made in USA. Serum samples were homogenized and used for determination of cholesterol
contents. The quantification of cholesterol was based on the experimental procedure of Dobreva
et al (2011).

Data Analysis
Data obtained were analysed using one-way analysis of variance (ANOVA). Duncan's multiple
range test, Tukey and LSD was used to separate the differences between mean values at P < 0.05.
All analyses were computed using SPSS package version 20.0. Data were presented as mean value
± standard deviation.
Results and discussion
The table 2 below shows the proximate composition of the experimental diets supplemented with
lemon oil. For the moisture content, there were no significant difference (p>0.05) among the values
for each increment in lemon oil with the highest value being 8.40%. The ash and lipid compositions
have their highest values (8.50% and 6.42%) at 1.0% lemon oil increments. There was also an
indication of a significance difference (P<0.05) among the values. The fibre content shows an
upward trend (P<0.05) in its value in relation to the control diet, with the control having a value
of 12.35% while the 1.2% increment of lemon oil has 12.50% fibre content. There was a fair
constant level in the values of the crude protein and the NFE. Their control diets have 40.10% and
26.74% respectively while the diet T5 has 40.20% and 25.80% respectively. There was also a
significant difference (P<0.05) among the values, indicating that they are very closely related. It
is implied that any significant changes in the growth pattern of the fishes and their nutritional value
is as a result of the supplementation with the lemon oil.

Table 2: Proximate composition of experimental Diets

Parameters T1(Control) T2(0.4%) T3(0.8%) T4(1.0%) T5(1.2%)
(%)

Moisture 7.75±0.08a 8.20±0.07a 8.10±0.38a 8.30±0.03a 8.40±0.00a

Ash 8.39±0.05a 8.18±0.05a 8.45±0.19a 8.50±0.02a 7.94±0.05a

Lipids 4.67±0.09a 5.31±0.07a 5.40±0.07a 5.42±0.08a 5.16±0.10a

Fibre 12.35±0.45a 12.43±0.40a 12.21±0.08a 12.37±0.86a 12.50±0.19a

Crude protein 40.10±0.19a 40.20±0.00a 40.06±0.01a 40.10±0.23a 40.20±0.24a

NFE 26.74±0.03a 25.68±0.42a 25.78±0.57a 25.70±0.70a 25.80±0.10a

*Figures presented are means and standard deviation (mean ± SD) for the experimental feed
in triplicate (n=15).

Proximate Composition of Experimental Fish

The table 3 below shows the results obtained from the proximate analysis of the fish fed with
lemon oil. It indicated that there was a significant difference (P<0.05) in the moisture content with
an average value of (8.22%) of the experimental fish in all the dietary treatments. This value is
higher than the control (7.99%) which shows an upward trend in its moisture content as a result of
the addition of lemon oil. Also, the ash and lipid contents showed an increasing and decreasing
(P<0.05) trend as the concentration of the lemon oil increases in the feeds with the control having
the highest value 12.78% and 15.49% for ash content while 15.65% and 14.88% for the lipid
content. However, it was observed that there was significant decrease (P<0.05) in the protein
content as the concentration of lemon oil increases across the dietary treatments. The control has
the highest value (60.36%) while the 1.2% inclusion level had the lowest value of 56.20% crude
protein content. However, the NFE composition experienced a sharp increase away from the
control value (3.22%) as the concentration of lemon oil increases. The highest value was at the
1.2% inclusion level (5.09%) while the mean value was 4.20% NFE content.

Table 3: Proximate composition of the experimental fish

Parameters (%) T1(Control) T2 (0.4%) T3 (0.8%) T4 (1.0%) T5 (1.2%)

Moisture 7.99±0.10a 8.22±0.31a 8.20±0.20a 8.10±0.24a 8.34±1.34a

Crude Protein 60.36±0.57a 60.10±0.11a 57.50±0.32a 59.20±0.20a 56.20±0.87a

Ash 12.78±0.31a 13.00±0.08a 14.30±0.15a 14.30±0.06a 15.49±0.02a

Lipid 15.65±0.43a 15.20±0.12a 15.88±0.35a 14.30±0.75a 14.88±0.27a

NFE 3.22±0.62a 3.48±0.36a 4.12±0.38a 4.10±0.24a 5.09±0.39a

*Values with different superscript in the same row indicate significant difference at P<0.05.
**Figures presented are means and standard deviation (mean ± SD) for fifteen fish from
three replicates (n = 15).

Growth Indices

At the end of the 56 days of feeding experiment, it was observed that the total weight gained of
the lemon oil supplemented feeds was significantly higher (P< 0.05) than the control group. The
treatment with the 1.2% inclusion level gained 14.10g from the 3.52g initial weight, while the
control group had an initial weight of 3.51g and gained 13.91g. This is a factor of the final weight
gained after the feeding experiments were over. However, the specific growth rate follows a
different pattern as the weight gain discussed earlier. The control had the highest growth rate of
2.50% as opposed to the highest inclusion level (1.2% lemon oil concentration) with a growth rate
of 2.46% while the average was 2.48%. For the feed conversion ratio, there was no significant
difference (P>0.05) among all the values for the dietary treatments. The diet T2 has the lowest
value (3.14%) and increases progressively to 3.45% for the 1.2% lemon oil inclusion level. The
feed efficiency rate was relatively stable and closely related, with the control having a 0.29% FER
rate and the feed T5 had 0.30% while the average FER was 0.30%. The survival rate for the fishes
fed with lemon oil shows no significance difference (P>0.05) among their values in relation to the
control. The survival rate increased as the concentration of lemon oil increases, with the control
having a 73.33% survival rate as opposed to 86.67% for the feed T2. This shows that the
probability of survival is higher in feeds supplemented with lemon oil. It also shows that dietary
oil improves the growth and nutritional utilization of fish.
Table 4: Growth and nutrient utilization of African catfish fed dietary lemon

Parameters T1 T2 T3 T4 T5

MIW 3.51±0.05a 3.51±0.02a 3.52±0.02a 3.50±0.02 a 3.52±0.06a

MFW 17.42±2.19a 17.30±0.32a 17.40±0.84a 17.15±0.68a 17.62±0.52a
MWG 13.91±2.14a 13.79±0.32a 13.88±0.83a 13.65±0.71a 13.48±0.48a
SGR 2.50±0.17a 2.49±0.04a 2.49±0.09a 2.48±0.11a 2.46±0.06a
FI/day 46.65±2.58a 46.50±0.55a 46.65±1.35a 46.50±1.16a 46.50±1.25a
FCR 3.35±0.07a 3.14±0.04a 3.36±0.02a 3.40±0.04a 3.45±0.06a
FER 0.29±0.03a 0.32±0.01a 0.30±0.01a 0.29±0.01a 0.30±0.02a
SURVIVAL 73.33±6.67a 86.67±6.67a 85.00±7.70a 84.44±12.37a 84.44±4.44a

*Values with different superscript in the same row indicate significant difference at P<0.05.
**Figures presented are means and standard deviation (mean ± SD) for fifteen fish from
three replicates (n = 15).

Mineral Composition of the Experimental Fish

The mineral composition of the fish as indicated in table 5 shows no significant different (P>0.05)
between the control and the various fish feed dietary lemon oil for the calcium composition.
Calcium and phosphorus contents showed an increasing trend as the lemon oil concentration
increases in the feeds. The T5 diets have the highest value (50.90 mg and 10.80 mg) and the lowest
(47.75 mg and 9.40 mg) while their average were 50.45 mg and 10.39 mg respectively. For
manganese, magnesium and zinc contents, there were significant increase (P<0.05) in the values
as the concentration level of the lemon oil increases in the dietary treatments. For magnesium, the
control had 38.15 mg while the T5 diet had 46.64 mg. Manganese had its control at 0.19 mg while
the feed T2 had the highest value of 0.33 mg while for zinc, the highest value was T5 with 2.60
mg zinc content. It shows that dietary oil improves the mineral composition in the fish.
Table 5: Mineral composition of the experimental fish (ppm)

Parameters T1 T2 T3 T4 T5

Calcium 47.75±1.75a 49.77±3.41a 50.32±1.38a 50.80±2.32a 50.90±1.74a

Magnesium 38.15±1.37a 38.95±0.78a 42.22±5.21ab 45.60±1.13b 46.64±1.13b

Manganese 0.19±0.02a 0.33±0.04b 0.24±0.04ab 0.25±0.03ab 0.25±0.03ab

Zinc 1.88±0.23a 1.89±0.18a 2.09±0.42b 2.16±0.34ab 2.60±0.11b

Phosphorus 9.40±0.75a 9.66±0.39a 10.37±1.76a 10.74±0.94a 10.80±0.66a

*Values with different superscript in the same row indicate significant difference at P<0.05.
**Figures presented are means and standard deviation (mean ± SD) for fifteen fish from
three replicates (n = 15).

Serum and Tissue Biochemistry

The 56 days of feeding experiment produced the results of cholesterol level as presented in figure
1 below. There was no statistical significant difference (P>0.05) among all the values with the
exception of feed T4. There was a decreasing trend of results as the concentration of lemon oil
increases across the feed treatments. The control had the highest value of 3.59mg/dl, while the
mean value was 3.26 mg/dl, which is lower than the control diet. It shows that dietary oil reduces
the cholesterol content of the fish.
Table 8 presents the results of an analysis conducted on the effects of using essential oil from
Lemon (Citrus limon) on the production of African Catfish (Clarias gariepinus). The table
presents the mean values of different parameters measured in five treatments (T1-T5) representing
inclusion levels of 0, 0.4, 0.8, 1.0, and 1.2g/100g respectively.
The parameters measured are AST (aspartate transaminase), TP (total protein), CAT (catalase),
SOD (superoxide dismutase), and LPO (lipid peroxidation). These parameters are often used as
indicators of the physiological and biochemical status of the fish.
Based on the inclusion levels of Citrus limon on fish feed provided, the results suggest that
increasing the inclusion level of Citrus limon in fish feed up to 1.2g/100g positively impacts the
physiological and biochemical status of African Catfish. The results show that the highest mean
values of AST, CAT, and SOD were observed in treatment T3, which had an inclusion level of
0.8g/100g citrus limon which indicates that the essential oil from Lemon has a positive effect on
the liver function and antioxidant defence system of the fish. Similarly, the values of SOD are
significantly higher in treatments T3 (0.8g/100g) and T5 (1.2g/100g), indicating an increase in the
antioxidant capacity of the fish. A study by Afaf. N. et al., (2019) had similar result, showed no
significant difference in the growth parameters of the fish
On the other hand, the values of TP are significantly lower in treatment T2 (0.40g/100g inclusion
level), which suggests a decrease in protein synthesis the values of total protein (TP) were
significantly lower compared to the other treatments, suggesting that higher inclusion levels of
citrus limon may be required to maintain normal protein synthesis in the fish. The values of LPO
are relatively low and show no significant differences among the treatments. This indicates that
the essential oil from Lemon has no adverse effect on the oxidative stress status of the fish.
Overall, the results suggests that the optimal inclusion level of Citrus limon in fish feed may be
around 0.8g/100g, as this resulted in the highest values of liver function and antioxidant defence
system parameters.
In summary, the results suggest that the essential oil from Lemon can be used as a dietary
supplement for African Catfish to improve their liver function, antioxidant and defence system.

Cholesterol Analysis of C. gariepinus fed C. limon

4
3.59 3.51
3.5 3.31
3.19
3.02
3
Cholesterol Level (mg/dl)

2.5

1.5

0.5

0
0.00% 0.40% 0.80% 1.00% 1.20%
Treatments (1 - 5)

Figue 1: Cholesterol analysis for C. gariepinus fed C. limon for 56 days

Table 8: Showing results of Serum biochemical analysis

PARAMETERS T1 T2 T3 T4 T5

AST 36.17+2.12a 45.00 + 2.29a 77.33+2.03b 70.67+6.09b 74.33 + 0.44b

TP 2.04+0.18 a 1.55 + 0.06 a 1.49+0.13 a 1.74+0.23 a 1.50 + 0.26 a
CAT 31.89+4.25 a 77.76+1.14 b 68.44+0.93 c 72.01+0.92 c 66.48 + 1.68c
SOD 62.16+1.09 a 60.26+5.95 a 76.73+0.45 b 73.40+3.07 a 80.00 + 2.30 b
LPO 2.3E-6 + 2.7E-7 7.5E-6+6.1E- 1.7E-6+14E- 2.7E-6+2.1E- 3.3E-6+2.9E-
a
7a 7a 7a 7a

Values with different superscripts across row shows significant difference at (p < 0.05).

Heamatology Profile

The haematological analysis of the catfish fed with dietary lemon oil is showed in table 6. It was
revealed that the all the values had no significant difference (P>0.05). The Haemoglobin, White
Blood Cell and the Red Blood Cell counts were decreasing as the concentration of the lemon oil
increases across the treatments. The highest RBC was observed to be 2.52×103m/cumm (control
diet) and the lowest was seen in the diet T3 (2.10×103 m/cumm). For the white blood cell, the diet
T5 had the lowest value (5.88 ×103 cells/cumm), the highest value was at the diet T3 (7.57 ×103
cells/cumm) while the average was 6.98×103 cells/cumm, just slightly higher than the control. The
haemoglobin has the highest value at the diet T5 (7.63g/dl) while the lowes was at the diet T3
(6.37g/dl). The values for the Packed Cell Value was very closely related with the lowest value of
19.00% and a mean value of 21.02%, which is slightly lower than the control. For the MCHC,
MCH and MCV, there were slight gradual increase as the concentration of lemon oil was increased
in the diet treatments, with their values closely related to one another. It goes on to indicate that
dietary oil has no effect on the haematological profile of the fish.
Table 6: Heamatology profile of C. gariepinus fed lemon supplement

Parameters T1 T2 T3 T4 T5

PCV (%) 22.70±3.84a 20.70±0.00a 19.0±2.52a 21.70±0.67a 22.70±1.20a

HB (g/dl) 7.60±1.31a 6.93±0.14a 6.37±0.84a 7.33±0.20a 7.63±0.35a

WBC
6.33×103±1.10a 7.53×103±0.69a 7.57×103±0.87a 6.92×103±0.19a 5.88×103±0.76a
(cells/cu mm)
RBC
(million/cu 2.52×103±0.41a 2.28×103±0.19a 2.10×103±0.25a 2.43×103±0.07a 2.48×103±0.14a
mm)

MCHC (g/dl) 33.50±0.11a 33.5±0.17a 33.50±0.18a 33.80±0.45a 33.60±0.31a

MCH
30.20±0.34a 30.40±0.10a 30.30±0.44a 30.20±0.48a 30.80±0.81a
(picograms)

MCV (fl) 90.10±0.87a 90.80±0.29a 90.50±1.33a 89.30±2.56a 91.50±0.22a

*Means with in the same row with similar superscript are not significantly different at
P<0.05. **Figures presented are means and standard deviation (mean ± SD) for fifteen fish
from three replicates (n = 15).

Water Quality Parameters

The table 7 below shows the water quality parameters for the experimental conditions for the
catfish treatment. It shows that the temperature (26.95oC) was kept constant throughout the
different diet inclusion levels, ensuring that the fishes were subjected to the same water
temperature all through. It was observed that the dissolved oxygen increases as the concentration
of the lemon oil increases, with the highest value being the diet T5 (4.88) while the lowest is the
control (T1). There was also a significant difference (P<0.05) among all the values recorded for
the dissolved oxygen. For the pH levels, the water was kept at a weaker alkalinity level at the
control (7.62). This continued to increase as the concentration of the lemon oil increases along the
experimental stages, with the highest value obtained from T3 (7.74) and the average pH being 7.70
(weak alkalinity). The conductivity of the water kept on fluctuating as the concentration of the
lemon oil continues to increase at no significant difference (P>0.05). The highest value recorded
was at T2 (445.00) while lowest was at T5 (425.83).
Table 7: Water Quality parameters

Parameters T1 T2 T3 T4 T5

Temperature 26.95±0.00a 26.95±0.00a 26.95±0.00a 26.95±0.00a 26.95±0.00a

DO2 4.80±0.88a 4.81±0.58a 4.83±0.27a 4.87±0.15a 4.88±0.30a

pH 7.62±0.02a 7.68±0.01a 7.74±0.07a 7.66±0.07a 7.71±0.19a

Conductivity 426.50±24.84a 445.00±7.11a 427.00±6.79a 408.17±9.70a 425.83±6.22a

*Values with different superscripts across rows shows significant difference

Conclusion

Essential oils (C. limon) are safe additives to food that promote fish growth in place of antibiotics
in animal husbandry. But in Nigeria, African catfish are the most heavily farmed fish and are linked
to disease outbreaks. Against raise production and achieve sustainable development, they thus
need well-formulated diets that would speed up growth, ensure optimum nutritional utilization in
the nourishment, and boost immunity against infection from a disease. The study investigated the
effects of using lemon essential oil in the diet of African catfish (Clarias gariepinus) and found
that there were significant differences in weight gain, specific growth rate, and feed conversion
ratio among the different dietary treatments. However, the mean values of most of the parameters
measured, including MIW, MFW, MWG, SGR, FI, FCR, and FER, were similar across all the
treatment groups.
The study is consistent with previous studies that have explored the use of whole plants or extracts
from plants to promote growth and immune response in fish. Bulfon et al. (2015) reported that the
inclusion of garlic in the diet of common carp (Cyprinus carpio) led to improved growth
performance and reduced the prevalence of bacterial infections. Van Hai (2015) also found that
the inclusion of extract from Rhizophora apiculata in the diet of tiger shrimp (Penaeus monodon)
led to improved growth performance and immune response. Abdel Rahman et al. (2018) reported
that the inclusion of propolis in the diet of Nile tilapia (Oreochromis niloticus) led to improved
growth performance and immune response.
The use of plant extracts in aquaculture is gaining attention as a potential solution to improve
growth performance and immune response in fish, particularly in the face of intensifying
aquaculture production.
The current results indicated that, the essential oil adopted for this study (C. limon) is a supplement
that could improve the non-specific immune response, enzymatic antioxidant capacity and disease
resistance of the African catfish. It has also established that lemon oil can improve the body's
defense developmental efficiency, and antioxidant defense of the African catfish. The essential oil
used in this study's diet preparation acts as a healthy bacteria by improving food digestion,
speeding up the trial fish's rate of ingesting nutrients, and improving their general wellness by
boosting white blood cell production to combat the obstacle cells. However, as evident from the
findings, the 1.0% supplementation of lemon oil with the fish diet was discovered to produce the
best and optimal results in terms of the overall growth and development of Clarias gariepinus.

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