INTERNATIONAL JOURNAL OF RESEARCH AND SCIENTIFIC INNOVATION (IJRSI)
ISSN No. 2321-2705 | DOI: 10.51244/IJRSI |Volume XII Issue IX September 2025
Page 3681
Efficacy of Bitter Leaf (Vernonia Amygdalina) Extract for Removal of
Egg Adhesiveness during Artificial Propagation of African Catfish
(Clarias Gariepinus, Burchell 1822)
Timothy Okiki Ojebuola*., Olabode Thomas Adebayo., Oluyemi Kazeem Gbadamosi
Federal University of Technology, Akure, School of Agriculture and Agricultural Technology,
Department of Fisheries and Aquaculture, Akure, Ondo State, Nigeria
DOI: https://doi.org/10.51244/IJRSI.2025.120800332
Received: 04 Sep 2025; Accepted: 11 Sep 2025; Published: 13 October 2025
ABSTRACT
This study evaluated the optimal immersion period and concentration of bitter leaf (Vernonia amygdalina)
extract for effectively removing egg adhesiveness in African catfish (C. gariepinus). Two males (1.3 kg) and
two females (1.4 kg) were selected as broodstock for induced breeding. Three concentrations of bitter leaf
extract (0.5%, 1.0%, and 1.5%) were tested at immersion durations of 30, 60, and 90 seconds. Tannic acid
(0.75 g/L) served as the reference de-adhesion agent, while water alone was used as the control. Each
treatment was conducted in triplicate. Data were analysed using one-way ANOVA, followed by Tukey’s
multiple range test, while third-order polynomial regression was applied to identify the most effective
concentration and immersion period. The findings showed no significant differences (p > 0.05) in non-
adhesive egg percentage and hatchability between eggs treated with bitter leaf extract and those treated with
tannic acid. However, the combination of 0.5% bitter leaf extract with a 30-second immersion period exhibited
the highest de-adhesion efficiency, resulting in 97.40% fertilisation, 95.07% non-adhesive eggs, and 90.09%
hatchability. The study concludes that bitter leaf extract is an effective, eco-friendly, and low-cost alternative
to synthetic agents such as tannic acid for removing egg adhesiveness in C. gariepinus. Its use at 0.5%
concentration with a short immersion period (30 seconds) is recommended for hatchery operations to enhance
seed production efficiency.
Keywords: Bitter leaf, Clarias gariepinus, Egg stickiness, Non-adhesiveness, Hatching.
INTRODUCTION
The artificial propagation of African catfish (Clarias gariepinus) is essential in aquaculture but faces
challenges due to the adhesive nature of their eggs (Kwikiriza et al., 2025). When released into the water, these
eggs tend to clump together, reducing fertilisation and hatching rates and increasing larval mortality (Ojebuola
et al. 2024). This adhesiveness poses a significant obstacle in hatcheries, necessitating effective solutions to
ensure higher survival and productivity rates.
Over the years, several physical and chemical methods have been employed to mitigate the adhesiveness of C.
gariepinus eggs. Solutions such as urea, mud, milk, kaolin, and tannins have been used, sometimes requiring
species-specific applications (Kareem et al., 2016). While tannin solutions are effective for some species like
pikeperch (Żarski et al., 2015), they can be costly and may not be readily available in rural aquaculture
settings. Enzymatic treatments, such as Alcalase, show promise but also face issues of cost and availability
(Kristan et al., 2017; Ljubobratović et al., 2018). Aloe Vera gel and water leaf extracts have been used with
immersion periods of five and one minute, respectively, for African catfish (Fawehinmi et al., 2019). Ojebuola
et al. (2024) also reported that 1% concentration of okra leaf extract with a one-minute immersion period
yielded the best results for Clarias gariepinus. However, these methods often require precise control over
treatment times and conditions, which can be challenging to maintain consistently.
Given these limitations, there is a need for more accessible, cost-effective, and reliable methods to address egg
adhesiveness in Clarias gariepinus. One promising solution is the use of bitter leaf (Vernonia amygdalina), an
INTERNATIONAL JOURNAL OF RESEARCH AND SCIENTIFIC INNOVATION (IJRSI)
ISSN No. 2321-2705 | DOI: 10.51244/IJRSI |Volume XII Issue IX September 2025
Page 3682
abundant and inexpensive plant native to tropical Africa, commonly found in Nigeria, Cameroon, and Ghana.
This bushy plant, which can grow up to three meters tall, is characterised by its dark green leaves with a bitter
taste. The leaves contain beneficial compounds, including antioxidants, polysaccharides, minerals, proteins,
enzymes, and vitamins (Nwachukwu et al., 2014).
According to Ogwu and Ikhajiagbe (2023), bitter leaf is traditionally used in many African cultures for its
medicinal properties. It is used to treat ailments such as malaria, diabetes, gastrointestinal disorders, and to
improve general health. Its antimicrobial and anti-inflammatory properties are well-documented, making it a
valuable resource in both traditional medicine and modern pharmacology.
Despite its widespread use in other applications, limited research exists on the use of bitter leaf extract for
removing egg adhesiveness in Clarias gariepinus. Usunobun and Ngozi (2016) found that saponins, tannins,
alkaloids, flavonoids, triterpenoids, steroids and cardiac glycosides were high in V. amygdalina. Given its
bioactive components and accessibility, it stands as a potential solution that could be both effective and
economically viable for fish farmers, particularly in rural areas. This study aims to explore the efficacy of
bitter leaf extract in removing egg adhesiveness and to determine the optimal concentrations and immersion
periods needed to enhance fertilisation and hatching rates.
By investigating the use of bitter leaf extract, this research seeks to provide an alternative that could improve
the efficiency of African catfish hatcheries. The goal is to develop a method that addresses the issue of egg
adhesiveness while aligning with the practical and economic constraints faced by many aquaculture operations.
This could ultimately lead to higher productivity and sustainability in the cultivation of Clarias gariepinus.
MATERIALS AND METHODS
Study Area and brood fish
The experiment was conducted at the Teaching and Research Fish Farm of the Federal University of
Technology, Akure, located in Obakekere, Akure. Two healthy male and two female C. gariepinus, weighing
approximately 1.3 kg and 1.4 kg, respectively, were procured from a reputable fish farm in Akure. The fish
were placed in separate holding tanks (40 × 30 × 35 cm³) supplied with aeration and acclimated for five days.
During this period, they were fed with a local commercial diet, which was withdrawn 24 hours before artificial
induction of ovulation.
Collection and Identification of Plant Material
Fresh bitter leaf plants were collected within the Teaching and Research Fish Farm, Department of Fisheries
and Aquaculture, The Federal University of Technology, Akure. It was identified as Vernonia amygdalina at
the Herbarium of the Department of Crop, Soil and Pest Management, The Federal University of Technology,
Akure.
Preparation of Bitter Leaf Extract
Fresh, healthy bitter leaves were selected, identified by their dark green colour, elliptical shape, and bitter taste
due to compounds such as sesquiterpene lactones and tannins, the latter being an active agent in egg de-
adhesion (Żarski et al., 2015). About 500 g of leaves were harvested, inspected to ensure they were free from
pests and diseases, and thoroughly washed under running water until the rinse water was clear.
The cleaned leaves were cut into pieces of about 2–3 cm to increase surface area and facilitate extraction. They
were then manually crushed and squeezed in a clean basin, releasing their liquid extract. The mixture of
crushed leaves and liquid was filtered through a 1 mm mesh hand net to separate the aqueous extract from leaf
residues. Filtration was repeated until sufficient extract was obtained. The resulting greenish extract was
collected in a clean basin.
The extract was transferred into a dry, airtight plastic container and kept at room temperature (≈25°C) in a
shaded, dry place. It was allowed to stand for 30 minutes so that fine particles could settle, after which the
INTERNATIONAL JOURNAL OF RESEARCH AND SCIENTIFIC INNOVATION (IJRSI)
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clear supernatant was carefully decanted for use. The extract was freshly prepared and applied on the same day
as egg stripping to maintain quality and effectiveness.
The greenish extract was prepared into percentages as follows:
0.5% = 0.5ml of Bitter leaf extract in 99.5ml of water.
1% = 1.0ml of Bitter leaf extract in 99ml of water.
1.5% = 1.5ml of Bitter leaf extract in 98.5ml of water.
Preparation of Tannic Acid Solution (Reference de-adhesion agent)
Tannic acid solution that served as a reference de-adhesion agent was prepared by diluting 0.75g of tannic acid
into one litre of water according to Żarski et al. (2015).
Water without any of the extracts served as the control.
Preparation of Spawning Bowls
Fifty-seven spawning bowls of 4 litres capacity were used for the experiment. The bowls were thoroughly
washed and dried. The bowls were labelled according to the inclusion levels of the treatments, tannic acid
solution (0.5%, 1% and 1.5%) and control as well as the immersion periods (30 seconds, 60 seconds and 90
seconds). The bowls were filled with 100ml of water (control), 99.5ml of water (0.5%), 99ml of water (1%)
and 98.5ml of water (1.5%) respectively.
Milt and Egg Collection
The female brooders were injected with hormone (Ovaprim Syndel Laboratories Ltd., Nanaimo, BC Canada
V9S 4M9) at an angle 45° with the needle pointing towards the gonad region. The injected brooder was kept
inside separate plastic tanks (24 x 12 x 12 cm
3
) containing water and tightly covered with a perforated lid.
After a latency period of 12 hours, slight pressure was applied to the abdominal cavity to express the eggs into
a clean bowl. The male testes were removed by abdominal dissection and cleaned with a towel; the milt was
gently squeezed out and collected in a beaker.
Fertilisation and immersion
Wet fertilisation was used in the experiment. Milt collected was then mixed with a small quantity of saline
solution. 1g of the striped eggs was carefully weighed on nylon, and each measured egg was fertilised with the
prepared milt (0.01ml of milt to 1g of eggs (FAO 1996). The eggs were randomly rinsed inside the spawning
bowls and subjected to the treatments.
Experimental Design
Each treatment triplicate received 1g of eggs (1g of eggs contained 700 eggs using a Metler balance, Model:
Toledo PB 8001).
The fertilised eggs were placed in three treatment concentrations of Bitter leaf extract, tannic acid solution
(reference de-adhesion agent) at (0.5, 1 and 1.5) %, and water (control). There were three replicates for each of
the inclusion levels. The exposure time was 30, 60 and 90 seconds, respectively, to determine the optimum
concentration and immersion period of bitter leaf extract. After the speculated exposure period, the
concentrated water was decanted, and then clean water was replaced to incubate the eggs in the spawning
bowls.
Evaluation of non-adhesive eggs, hatchability, survival and deformity indices
To determine the efficacy and efficiency of bitter leaf extract in removing egg adhesiveness, the parameters
INTERNATIONAL JOURNAL OF RESEARCH AND SCIENTIFIC INNOVATION (IJRSI)
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assessed were computed according to the method described by Adebayo (2006).
Non-adhesive eggs (%) = number of non − adhesive egg initial number of eggs
⁄
× 100
Hatchability (%) = (Number of eggs hatched) ⁄ (Total number of eggs incubated) × 100
𝐒𝐮𝐫𝐯𝐢𝐯𝐚𝐥
(
%
)
= number of hatchling at 72h Total number of hatchling at 0h
⁄
× 100
Hatching index: For each treatment, a hatching index expressed as the percent viable embryos at 72 h post-
hatch/initial number of eggs was calculated. This index represented the percentage (%) of the hatched larvae
obtained from the initial number of eggs.
Deformity (%) = (Number of deformed larvae) ⁄ (Total number of larvae 72 h post-hatch) × 100
Larvae Size: The mean total larval length at 72 h post-hatch was calculated using ImageJ 1.34 software
(Rasband 1997–2011) as described by Ben Khemis et al. (2014).
Water Quality Parameters
Water quality parameters such as temperature, pH and dissolved oxygen concentration were monitored twice
throughout the study period using a mercury-in-glass thermometer(YSI-DO 550, U.S.A), a pH meter (Hanna
H198106 model) and a dissolved oxygen meter (JPP-607 model) as described by APHA (1987).
Statistical analysis
All percentage data at different concentrations and immersion periods were subjected to an Analysis of
Variance test. Also, Tukey's Honestly Significant Difference test was used as a follow-up procedure.
Polynomial regression analysis was then used to determine the best concentration and immersion period of
biter leaf extract treatment that effectively removed egg adhesiveness. All analysis was performed at a 0.05
significance level.
RESULTS
Effects of bitter leaf extract on Clarias gariepinus eggs
Adhesiveness of eggs of C. gariepinus exposed to varying concentrations and immersion periods of Bitter
leaf extract
The results of egg adhesiveness in C. gariepinus exposed to different concentrations and immersion periods of
bitter leaf extract are presented in Table 1. The highest percentage of non-adhesive eggs (95.07%) was
recorded at 0.5% concentration with a 30-second immersion period, while the lowest (73.41%) occurred at
1.5% concentration with a 90-second immersion period. Similarly, eggs treated with tannic acid solution
showed non-adhesive values ranging from 92.52% at 0.5% concentration with a 60-second immersion period
to 80.34% at 1.5% concentration with a 90-second immersion. In contrast, the control group recorded only
25.62% non-adhesive eggs. Statistical analysis revealed no significant difference (p > 0.05) between bitter leaf
extract and tannic acid treatments, but both differed significantly from the control. Generally, egg detachment
decreased in both bitter leaf and tannic acid solutions as concentration and immersion periods increased.
Figure 1 presents the highest percentage of non-adhesive C. gariepinus eggs treated with 0.5% bitter leaf
extract at different immersion times, compared with tannic acid as the reference agent.
INTERNATIONAL JOURNAL OF RESEARCH AND SCIENTIFIC INNOVATION (IJRSI)
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Figure 1: Egg adhesiveness of C. gariepinus treated with 0.5% bitter leaf extract at varying immersion times.
Incubation period of C. gariepinus exposed to varying concentrations and immersion periods of Bitter
leaf extract
The incubation period of eggs treated with bitter leaf extract ranged from 23 hours 50 minutes at 1%
concentration (30 seconds) to 24 hours 50 minutes at 1.5% concentration (90 seconds), as shown in Table 1.
For eggs treated with tannic acid solution, incubation time varied from 23 hours 53 minutes at 0.5%
concentration (30 seconds) to 24 hours 65 minutes at 1.5% concentration (90 seconds). In contrast, eggs
incubated in water (control) recorded the longest incubation period of 25 hours 73 minutes. Statistical analysis
showed no significant difference (p > 0.05) between incubation periods of eggs immersed in bitter leaf extract
and tannic acid solution, though both were shorter than the control.
Percentage hatchability of C. gariepinus exposed to varying concentrations and immersion periods of
Bitter leaf extract
The percentage hatchability decreased with increasing concentrations of bitter leaf extract, as presented in
Table 1. The highest hatchability (90.09%) was obtained in eggs treated with 0.5% bitter leaf extract for 30
seconds, while eggs exposed to 0.5% tannic acid solution for 30 seconds recorded a hatchability of 85.07%.
Hatchability values obtained from treatments with 0.5% bitter leaf extract and tannic acid solution were high
and not significantly different (p > 0.05). However, both were significantly higher (p < 0.05) than the control
group, which recorded the lowest hatchability of 43.24%. Figure 2 presents the highest percentage hatchability
of C. gariepinus treated with 0.5% bitter leaf extract at different immersion times, compared with tannic acid
as the reference agent.
Percentage hatching index of C. gariepinus exposed to varying concentrations and immersion periods of
Bitter leaf extract
The percentage hatching index decreased with increasing concentrations of bitter leaf extract, corresponding
with the observed hatchability trends as presented in Table 1. The lowest hatching index (9.55%) was recorded
in the control group, while the highest value (72.60%) occurred in eggs exposed to 0.5% bitter leaf extract for
30 seconds. In comparison, eggs treated with 0.5% tannic acid solution for 30 seconds achieved a hatching
index of 57.14%. Notably, the hatching index observed at the lowest concentration of bitter leaf extract (0.5%
with 30 seconds immersion) was significantly higher (p < 0.05) than that of the control and other treatments
across varying concentrations and immersion periods.
25.62
95.07
88.86
86.76
92.52
77.91
86.62
0
20
40
60
80
100
120
0.5% Bitterleaf (BL) and Tannic Acid (TA)
Non-adhesive eggs (%)
INTERNATIONAL JOURNAL OF RESEARCH AND SCIENTIFIC INNOVATION (IJRSI)
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Figure 2: Percentage Hatchability of C. gariepinus treated with 0.5% bitter leaf extract at varying immersion
times.
Deformed larvae of C. gariepinus exposed to varying concentrations and immersion periods of Bitter
leaf extract
No deformity of larvae was observed in this experiment. The survived larvae were very active and responsive
to feeding.
Percentage survival of C. gariepinus exposed to varying concentrations and immersion periods of Bitter
leaf extract
The survival larvae percentage showed that survival decreased as concentration and immersion period
increased, as shown in Table 1. 80.90% was the highest, and this was observed in 0.5% concentration with a
30-second immersion period, and the lowest (51.30%) was in 1.5% concentration (90 seconds) of bitter leaf
extract. Also, 67.41% was the highest and this was observed in 0.5% concentration with 30 seconds immersion
period and lowest (54.89%) in 1.5% concentration (90 seconds) of tannic acid solution while survival of
hatched larvae from the control group was 22.11% which was the least when compared with survived larvae
exposed to varying concentrations and immersion periods of bitter leaf extract and tannic acid solution.
However, there was a significant difference (p<0.05) between the highest larvae survival (80.90%) recorded in
0.5% concentration with a 30-second immersion period of bitter leaf extract and other rinsing agents at varying
concentrations and immersion periods, including the control.
Larvae size of C. gariepinus exposed to varying concentrations and immersion periods of Bitter leaf
extract
The result of larvae size of C. gariepinus exposed to varying concentrations and immersion periods of bitter
leaf extract is shown in Table 1. The larvae size of C. gariepinus obtained for eggs immersed in bitter leaf
extract ranged from 0.32mm to 0.38mm in 1.5% (90 seconds) and 0.5% (30 seconds), respectively. Also,
larvae size of C. gariepinus obtained for eggs immersed in tannic acid solution varied between 0.29mm to
0.35mm in 1.5% (90 seconds) and 0.5% (30 seconds), respectively while 0.23mm was recorded in the control,
which is the smallest size compared to other larvae sizes of those immersed in varying concentrations and
immersion periods. Hence, there was no significant difference (P>0.05) in larvae size between concentrations
and immersion periods of eggs immersed in bitter leaf extract and tannic acid solution but there was significant
different (P<0.05) when compared with the control.
43.24
90.09
85.07
82.84
75.66
75.4
72.17
0
20
40
60
80
100
120
0.5% Bitterleaf (BL) and Tannic Acid (TA)
(%) Hatchability
INTERNATIONAL JOURNAL OF RESEARCH AND SCIENTIFIC INNOVATION (IJRSI)
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Table 1: Percentages of egg adhesiveness, fertility, hatchability, survival and incubation period of bitter leaf
extract, tannic acid solution and water.
Rinsing
Agents
Concentrat
ion (%)
Immersion
time(sec)
Non-
adhesive
eggs (%)
Incubation
period
(mins)
Hatching
(%)
Survival (%)
Larvae size
(mm)
Water
(Control)
25.62±0.10
b
1544±2.90
b
43.24±0.17
b
22.11±0.09
c
0.23±0.01
b
Bitter leaf
0.5
30
95.07±0.36
a
1411±2.65
a
90.09±0.34
a
80.90±0.31
a
0.38±0.01
a
60
86.76±0.33
a
1415±2.66
a
82.84±0.31
a
68.14±0.26
b
0.37±0.01
a
90
77.91±0.30
a
1416±2.66
a
75.40±0.29
a
57.89±0.22
b
0.36±0.01
a
Tannic Acid
0.5
30
88.86±0.34
a
1412±2.65
a
85.07±0.33
a
67.41±0.26
b
0.35±0.01
a
60
92.52±0.35
a
1413±2.65
a
75.66±0.29
a
67.12±0.26
b
0.36±0.01
a
90
86.62±0.33
a
1421±2.67
a
72.17±0.27
a
56.15±0.20
b
0.34±0.01
a
Bitter leaf
1
30
89.60±0.34
a
1410±2.65
a
82.77±0.31
a
76.93±0.29
a
0.36±0.01
a
60
81.42±0.31
a
1413±2.66
a
71.80±0.27
a
63.31±0.24
b
0.35±0.01
a
90
73.73±0.28
a
1421±2.67
a
69.43±0.26
a
51.53±0.20
b
0.33±0.01
a
Tannic Acid
1
30
86.35±0.33
a
1413±2.66
a
81.44±0.31
a
65.28±0.25
b
0.33±0.01
a
60
86.57±0.33
a
1415±2.66
a
72.25±0.27
a
62.37±0.24
b
0.32±0.02
a
90
82.86±0.31
a
1416±2.66
a
71.77±0.27
a
62.33±0.24
b
0.31±0.02
a
Bitter leaf
1.5
30
85.23±0.32
a
1419±2.67
a
78.20±0.30
a
70.18±0.27
a
0.35±0.01
a
60
78.03±0.30
a
1421±2.67
a
64.50±0.25
b
59.74±0.23
b
0.34±0.01
a
90
73.41±0.28
a
1470±2.77
a
60.78±0.23
b
51.30±0.20
b
0.32±0.01
a
Tannic Acid
1.5
30
82.18±0.31
a
1421±2.67
a
71.77±0.27
a
56.96±0.22
b
0.33±0.01
a
60
80.53±0.31
a
1454±2.74
a
72.04±0.00
a
56.64±0.22
b
0.31±0.02
a
90
80.34±0.31
a
1479±2.78
a
69.77±0.00
a
54.89±0.21
b
0.29±0.01
ab
The mean values in the same column with different superscript were significantly different (P<0.05)
Water quality parameters of varying concentrations and immersion periods of Bitter leaf extract
The water quality parameters of eggs immersed in bitter leaf extract are presented in Table 2. The lowest
temperature (27.02°C) was recorded at 0.5% concentration with a 30-second immersion period, while the
highest temperature (27.18°C) occurred at 1% and 1.5% concentrations with a 90-second immersion period.
The lowest pH (7.05) was observed at 0.5% concentration with a 30-second immersion period, whereas the
highest pH (7.18) was recorded at 1% concentration with a 90-second immersion period. Dissolved oxygen
ranged from a minimum of 5.50 mg/L at 1.5% concentration with a 90-second immersion period to a
maximum of 6.05 mg/L at 0.5% concentration with a 60-second immersion period. All results obtained were
not significantly different (P > 0.05) from those recorded in the tannic acid solution. Similarly, the water
quality parameters of eggs immersed in bitter leaf extract, tannic acid solution, and the control showed no
INTERNATIONAL JOURNAL OF RESEARCH AND SCIENTIFIC INNOVATION (IJRSI)
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significant differences (P > 0.05). Overall, the monitored parameters remained within suitable ranges to
support fish growth across all treatments.
The optimum concentration of bitter leaf extract used as a de-adhesive agent during artificial
propagation of C. gariepinus
At the end of the experimental trial, the optimum concentration that can efficiently remove egg adhesiveness in
C. gariepinus using bitter leaf extract was observed at a concentration of 0.65% using 3rd order polynomial
regression, as shown in Figure 3
Table 2: Physico-chemical parameters of test solutions of varying concentrations and immersion periods of
Bitter leaf
Rinsing agents
Concentrat
ion (%)
Immersion
time(mins)
Temperature
pH
DO
Water (Control)
27.00±0.99
a
7.17±0.04
a
6.05±1.20
a
Bitter leaf
0.5
30
27.02±1.01
a
7.05±0.02
a
5.70±0.71
a
60
27.08±0.89
a
7.08±0.03
a
6.05±1.13
a
90
27.09±1.08
a
7.14±0.02
a
5.82±0.54
a
Tannic Acid (Reference
de-adhesion agent)
0.5
30
27.04±1.00
a
7.08±0.04
a
5.65±0.64
a
60
27.10±0.96
a
7.16±0.04
a
5.75±0.78
a
90
27.12±0.95
a
7.18±0.03
a
5.70±0.72
a
Bitter leaf
1
30
27.12±0.04
a
7.12±0.04
a
5.61±0.57
a
60
27.17±0.01
a
7.17±0.01
a
5.66±0.50
a
90
27.18±0.04
a
7.18±0.04
a
5.75±0.77
a
Tannic Acid (Reference
de-adhesion agent)
1
30
27.09±1.09
a
7.11±0.04
a
5.65±0.78
a
60
27.17±0.90
a
7.18±0.06
a
5.60±0.57
a
90
27.10±0.94
a
7.19±0.04
a
5.65±0.79
a
Bitter leaf
1.5
30
27.17±1.05
a
7.10±0.06
a
5.75±0.64
a
60
27.10±0.94
a
7.12±0.04
a
5.85±0.92
a
90
27.18±0.95
a
7.13±0.03
a
5.50±0.71
a
Tannic Acid (Reference
de-adhesion agent)
1.5
30
27.06±0.94
a
7.14±0.04
a
5.65±0.50
a
60
27.11±1.00
a
7.18±0.05
a
5.95±1.20
a
90
27.13±0.80
a
7.21±0.01
a
6.00±1.27
a
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The mean values in the same column were not significantly different (P > 0.05).
Fig. 3: The optimum concentration of bitter leaf extract used as a de-adhesive agent during artificial
propagation of C. gariepinus
DISCUSSION
Effects of bitter leaf extract on Clarias gariepinus eggs.
Adhesiveness of eggs of C. gariepinus exposed to varying concentrations and immersion periods of bitter
leaf extract
Clarias gariepinus eggs immersed in bitter leaf extract, which contains tannins as the main active compound,
achieved 95.07% egg detachment. The highest effectiveness was observed at the lowest concentration (0.5%)
with the shortest immersion period (30 seconds). Tannins are polyphenolic compounds known to precipitate
proteins and disrupt mucopolysaccharides in the egg’s adhesive layer, thereby reducing stickiness and enabling
easier separation (Salisu et al., 2021). This biochemical action likely explains the rapid and efficient de-
adhesion observed in the present study.
The result compares favourably with Fawehinmi et al. (2019), who reported 93.77% detachment using
waterleaf extract containing tannic acid at 1% concentration with a 1-minute immersion. It also aligns with
Asraf et al. (2013), who identified one minute as the optimal rinsing period for African catfish eggs using urea,
and with Żarski et al. (2015), who reported high detachment rates (86.5% and 80.5%) in eggs treated with
tannic acid for 1–2 minutes. However, these results contrast with Demska-Zakes et al. (2005), who found that
low tannic acid concentrations combined with short immersion periods were ineffective, possibly due to
insufficient exposure to initiate protein precipitation.
However, the findings suggest that bitter leaf extract is an effective natural de-adhesion agent, offering optimal
fertilisation and minimal egg clumping at just 0.5% concentration with a 30-second immersion. Prolonged
exposure or higher concentrations may reduce efficiency, likely due to excessive protein precipitation leading
to damage or over-hardening of the chorion.
Incubation period of C. gariepinus exposed to varying concentrations and immersion periods of bitter
leaf extract
The incubation periods of eggs exposed to bitter leaf extract did not differ significantly across treatments.
y = 102.96x
3
- 309.96x
2
+ 268.14x + 25.62
R² = 1
0
20
40
60
80
100
120
0 0.5 1 1.5 2
30 sec
60 sec
90 sec
Poly. (30 sec)
Egg detachment
(%)
Concentration (%)
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According to SRAC (2006), incubation duration is strongly influenced by temperature and exposure period. In
this study, the first hatching was observed at 23 hours 50 minutes in eggs treated with 1% bitter leaf extract for
30 seconds. This finding aligns with Adebayo and Olayinka (2009), who reported the first hatching at 24.5
hours under the lowest formalin concentration, noting that longer exposure of C. gariepinus eggs to formalin
increased hatching time. Similarly, Ayoola et al. (2012) reported an incubation range of 21–26 hours, which
corroborates the present results.
Furthermore, incubation time in this study appeared unaffected by the relatively short immersion period. The
recorded water temperature (27.00–27.18°C) falls within the optimal range of 23.89–29.44°C reported by
Adebayo (2006) for C. gariepinus hatching, confirming that suitable thermal conditions supported normal
embryonic development.
Percentage hatchability of C. gariepinus exposed to varying concentrations and immersion periods of
bitter leaf extract
The eggs immersed in 0.5% bitter leaf extract for 30 seconds achieved the highest hatchability of 90.09%. This
is consistent with Żarski et al. (2015), who reported peak hatching rates of 95% in pikeperch eggs treated with
tannin solution for 1 minute. Similar results were reported by Thai and Ngo (2004), who achieved 86.3%
hatchability using pineapple juice, while salt/urea/tannin at 1% concentration produced 70.2%. Fawehinmi et
al. (2019) also documented about 70% hatchability in eggs immersed in waterleaf extract for 1 minute.
The superior performance of bitter leaf extract can be linked to its tannin content, which effectively removes
the adhesive mucopolysaccharide coating while minimising prolonged chemical stress on the eggs. By
breaking down the sticky layer quickly at a mild concentration, tannins ensure better oxygen circulation and
nutrient absorption during incubation, which are critical for embryo development. This explains why
hatchability peaked at the lowest concentration with the shortest immersion time.
These findings reinforce the importance of optimising both concentration and exposure time. They agree with
Żarski et al. (2015), who stressed that the shortest possible immersion at the lowest effective concentration
maximises hatching outcomes when using tannic acid. Likewise, Asraf et al. (2013) reported that a 1-minute
rinsing period yielded high fertilisation and hatchability with minimal egg clumping, supporting the
effectiveness of mild but timely treatment.
Percentage hatching index of C. gariepinus exposed to varying concentrations and immersion periods of
bitter leaf extract
A significantly higher hatching index (P < 0.05) of 72.60% was obtained in the group treated with 0.5% bitter
leaf extract for 30 seconds. Similarly, Zarski et al. (2015) reported the highest hatching index in groups
exposed to 1–2 minutes of immersion in tannic acid. The hatching index represents the percentage of larvae
hatched relative to the initial number of eggs incubated, thereby providing a reliable measure of the actual
production efficiency of C. gariepinus larvae from the total eggs used.
Deformed larvae of C. gariepinus exposed to varying concentrations and immersion periods of bitter leaf
extract
No deformities were observed in the larvae throughout the experiment. The surviving larvae were highly active
and exhibited strong feeding responses. This finding is consistent with the report of Zarski et al. (2015), who
noted that immersion period and duration did not significantly affect deformity rates in hatched larvae treated
with tannic acid.
Percentage survival of C. gariepinus exposed to varying concentrations and immersion periods of bitter
leaf extract
The highest survival rate (80.90%) was observed in eggs exposed to the lowest concentration of bitter leaf
extract (0.5%) with a 30-second immersion period, while survival declined progressively with increasing
extract concentrations. This outcome aligns with the findings of Akpoilih and Adebayo (2010), who reported
INTERNATIONAL JOURNAL OF RESEARCH AND SCIENTIFIC INNOVATION (IJRSI)
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reduced survival rates with higher concentrations of formalin. Similarly, Ljubobratović et al. (2018)
documented embryo survival rates of 82.5% and 87.7% in Alcalase-treated eggs and those treated with milk
and kaolin, respectively, which corroborates the present study.
Larvae size of C. gariepinus exposed to varying concentrations and immersion periods of bitter leaf
extract
The eggs immersed in 0.5% bitter leaf extract for 30 seconds produced the highest larval size (0.38 mm),
whereas higher concentrations resulted in reduced larval size. This finding is consistent with Hakim et al.
(2008), who reported increased larval growth in common carp at lower salinity levels. Conversely, Demska-
Zakęś et al. (2005) highlighted that prolonged immersion of eggs in rinsing agents such as tannic acid could
decrease egg size or even cause disruption due to osmotic pressure.
Water quality parameters of varying concentrations and immersion periods of bitter leaf extract
Water quality parameters play a crucial role in the growth, development, and survival of different stages of
fish. Thus, determining the optimal range of these variables is essential for successful aquaculture practices
(Marimuthu et al., 2019). In this study, the recorded water temperature ranged between 27.18°C and 27.23°C.
This finding aligns with Adebayo (2006), who reported an optimal hatching temperature range of 23–29°C for
Clarias gariepinus. Similarly, Viveen et al. (1986) and Amaechi and Solomon (2015) suggested suitable
temperature ranges of 20–30°C and 26–27°C, respectively, for C. gariepinus larvae, which are consistent with
the present observations.
Water pH is another critical parameter influencing fish physiology, particularly in maintaining homeostasis
(Marimuthu et al., 2019). In the current study, pH values ranged from 7.05 to 7.21. These values are within the
recommended range of 6.7–7.5 suggested by Marimuthu et al. (2019) for optimal hatching and larval survival
of African catfish. Furthermore, Santhosh and Singh (2007) reported a broader suitable pH range of 6.7–9.5 for
fish breeding, which also corresponds with the present findings.
Dissolved oxygen (DO) is equally vital for fish development, as it influences both embryonic and larval
survival. The present study recorded DO levels between 5.50 mg/L and 6.05 mg/L. These values fall within the
range of 4.5–8.0 mg/L reported by Bhatnagar and Sangwan (2009) as suitable for fish breeding, thereby
confirming the adequacy of the water quality conditions during the experiment.
CONCLUSION AND RECOMMENDATION
This study established that 0.5% bitter leaf (Vernonia amygdalina) extract with a 30-second immersion period
produced the best results in reducing egg adhesiveness, improving hatchability, and enhancing larval survival
of Clarias gariepinus. While tannic acid solution at 0.5% concentration with a 60-second immersion period
showed similar effectiveness, its higher cost makes it less feasible for routine hatchery application.
The findings highlight bitter leaf extract as an effective, affordable, and easily prepared natural alternative for
egg de-adhesion. Its wide availability and low cost make it particularly suitable for small- and medium-scale
hatchery operators who often face resource constraints. Adoption at a larger scale could help reduce
dependence on synthetic chemicals, lower production costs, and improve seed availability for farmers.
From a sustainability perspective, the use of bitter leaf extract offers additional advantages. As a
biodegradable, plant-based material, it presents minimal risk of harmful residues entering aquatic systems,
supporting environmentally friendly aquaculture practices. Its preparation requires no specialised equipment,
which increases its practicality and promotes ease of adoption across different production systems, including
rural hatcheries.
It is therefore recommended that hatchery operators adopt bitter leaf extract at a 0.5% concentration with a 30-
second immersion period for egg de-adhesion. Further research should, however, explore large-scale
applications, long-term environmental effects, and possible integration with other hatchery management
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practices. By investigating these dimensions more deeply, bitter leaf extract has the potential to contribute not
only to improved hatchery efficiency but also to the broader goal of sustainable aquaculture development.
Statements And Declarations
Acknowledgement
The authors express their sincere appreciation to the people who gave their advice and support. Special thanks
to the staff of Teaching and Research Fish Farm; Department of Fisheries and Aquaculture Technology,
Federal University of Technology, Akure.
Competing Interest
Authors have declared that no competing interests exist. The authors declare that they have no known
competing financial or non-financial, professional, or personal conflicts that could have appeared to influence
the work reported in this paper.
Author Contributions
This work was carried out in collaboration among all authors. First Author designed the study, managed the
literature searches and wrote the first draft of the manuscript. The second Author wrote the protocol. The third
Author performed the statistical analysis, managed the analyses of the study, wrote the review and edited. All
authors read and approved the final manuscript.
FUNDING
The authors declare that no funds, grants, or other support were received during the preparation of this
manuscript”. This research work was funded by the authors
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