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International Journal of Fisheries and Aquatic Studies 2019; 7(3): 06-13
E-ISSN: 2347-5129
P-ISSN: 2394-0506
(ICV-Poland) Impact Value: 5.62
(GIF) Impact Factor: 0.549
IJFAS 2019; 7(3): 06-13
© 2019 IJFAS
www.fisheriesjournal.com
Received: 02-03-2019
Accepted: 05-04-2019
Bimlendu Kumar Mishra
Department of Zoology,
Trichandra Multiple Campus
Saraswati-Sadan Tribhuvan
University, Kathmandu, Nepal
Monowar Alam Khalid
Prof. Head, Member, World
Commission on Protected Areas,
IUCN, Department of
Environment Science, Integral
University, Kursi Road
Lucknow, Uttar Pradesh, India
Shyam Narayan Labh
Prof. Head, (RMC), Fulbright
Fellow and Visiting Professor,
University of Idaho, USA
Department of Zoology, Amrit
Campus, Tribhuvan University,
Thamel, Kathmandu, Nepal
Correspondence
Shyam Narayan Labh
Prof. Head, (RMC), Fulbright
Fellow and Visiting Professor,
University of Idaho, USA
Department of Zoology, Amrit
Campus, Tribhuvan University,
Thamel, Kathmandu, Nepal
Assessment of the effect of water temperature on length
gain, feed conversion ratio (FCR) and protein profile in
brain of Labeo rohita (Hamilton 1822) fed Nigella sativa
incorporated diets
Bimlendu Kumar Mishra, Monowar Alam Khalid and Shyam Narayan
Labh
Abstract
Temperature is one of the environmental factors with greatest influence on the growth performance of
animals. Thus, a 12 week study was conducted to investigate the effects of different water temperature on
length gain, feed conversion ratio and protein profile in brain of Indian major carp rohu (Labeo rohita
Hamilton 1822) in the wet laboratory of Fisheries Development and Training Center, Janakpurdham,
Nepal. Altogether six treatments (T1, T2, T3, T4, T5 and T6) of varied temperaturesas18-20 °C, 20-22 °C,
22-24 °C, 24-26 °C, 26-28 °C, 28-30 °C were fixed with the help of aquarium thermometer in 18 aquaria
(three replicates). Natural climatic condition was at that time around 17-18 °C hence in T1 tank 18 °C
was fixed in the beginning and after a month it was changed to 19 °C and in final month the temperature
was fixed as 20°C, thus this tank was considered as control. Similar temperature variations were applied
in other treated tanks and after 90 days of feeding final sampling were done. Nigella sativa (Kalaunji) a
medicinal plant seed was prepared using ethanol extract of Kalaunji along with other ingredients and fed
at the rate of 3% of fish body weight wet twice a day. Labeo rohita attained significantly higher body
length (10.59±0.06 cm) and length gain % (151.28±0.68) under water temperature range of 24-26 °C fed
in fish group of T4 aquaria. The fish reared in water having temperature between 24-26 °C showed cent
per cent survival rate and the best FCR value (1.18±0.13). Similarly, total protein in brain (15.98±0.41g)
was significantly higher in T4 group under water temperature range of 24-26 °C. Thus, it has been
concluded that water temperature ranging from 24-26 °C seemed to be the most effective for rearing of
Labeo rohita proved for better growth and healthy carp. Aquafarmers can be benefitted by using this
technique.
Keywords: Water temperature, length gain, FCR, Labeo rohita, Nigella sativa, brain
1. Introduction
Increase in water temperature has a direct impact on aquaculture. Fish and other aquatic
animals are susceptible to changing environment. Increase in the temperature affects
physiological processes, such as metabolism, growth, reproduction etc. which results in a loss
in the productivity [1, 2]. Fish survives well in the optimal temperature in between 15-25 0C [3].
Different fish species are resistant to different temperatures, beyond the particular temperature,
it becomes harmful for growth and damage physiological processes [4]. Temperature has
multiple effects on fishes, such as controlling, masking, limiting, directing, and as a lethal
agent, i.e., too much or too little can destroy the integrity of a fish causing its death [5]. Some
fish which shows physiological adaptations can survive well in changing environment [6].
Rohu (Labeo rohita Hamilton 1822) is the most important among the three Indian major carp
species used in carp polyculture systems [7]. This graceful Indo-Gangetic riverine species is the
natural inhabitant of the riverine system of northern and central India, and the rivers of Nepal,
Pakistan, Bangladesh and Myanmar. In India, it is almost present all rivers [8]. The species has
also been introduced in many other countries, including Sri Lanka, the former USSR, Japan,
China, Philippines, Malaysia, and some countries of Africa. Its growth rate shows reduction
with decrease in the temperature [9]. Nigella sativa, commonly known as black cumin seed is
an important herb belongs to family Ranuculaceae has a great medical importance. A number
of researchers have used black cumin seeds as enhancer for performance, growth and immune
system of some fish species.
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International Journal of Fisheries and Aquatic Studies
Keeping in view the information given above, it can be
envisaged that by understanding how temperature affects the
performance of fish, particularly during winter season, a
farmer can maximize his profit by exploiting maximum
production potential of local fish species. However,
information regarding the effect of water temperature on
various species of fish in Nepal is limited. Therefore, an
experiment was conducted to investigate the effect of
different temperature ranges on the growth performance and
feed conversion ratio of Labeo rohita.
2. Materials and Methods
2.1 Preparation of Kalaunji seeds incorporated diet
Kalaunji Nigella sativa (Family Ranunculaceae) is a widely
used medicinal plant. The seeds of N. sativa have been widely
used in the treatment of different diseases and ailments. In
Islamic literature, it is considered as one of the greatest forms
of healing medicine. It has been recommended for using on
regular basis in Tibb-e-Nabwi (Prophetic Medicine). It has
been widely used as antihypertensive, liver tonics, diuretics,
digestive, anti-diarrheal, appetite stimulant, and analgesics,
anti-bacterial and in skin disorders. Hence, feed was prepared
using extract of Kalaunji along with other ingredients and fed
at the rate of 3% of fish body weight wet twice a day at 9 am
and 4 pm.
Fig 1: Showing encapsulation of Kalaunji seeds in garden
Fig 2: Flowers of Kalaunji showing multi-health properties.
Fig 3: Refined seeds ready for extractions
As described in Labh et al. [10] with slight modifications;
black seeds of Kalaunji collected from Kathmandu local
market were identified by National Herbarium and Plant
laboratory, Department of Plant Resources, Ministry of
Forests and Soil Conservation, Government of Nepal. After
identification, the samples were transported to the Laboratory
of Fish Nutrition, Department of Zoology, Amrit Campus,
Tribhuvan University, and Kathmandu, Nepal. The crude
extracts were prepared separately by using ethanol (80%) and
distilled water. The seeds were properly washed with clean
tap water and spread over plain sheet of paper for air drying
under shade. After drying, the samples were powdered using
warring blender. A known quantity (20 g) of Kalaunji powder
was taken in a 250 ml conical flask and added with 100 ml of
ethanol (80%).
The ethanol with Kalaunji powdered mixtures were kept for
48 hrs on orbital shaker and then, the extract was filtered
using muslin cloth to exclude the residues. The extraction was
repeated two times, the filtrate pulled together was
centrifuged at 10,000 x g for 5 min. and the supernatant was
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International Journal of Fisheries and Aquatic Studies
collected. Further, the supernatant was concentrated using
water bath at temperature of 70 °C. A greasy final crude
material obtained was transferred to screw-cap bottle, labeled
and stored under refrigerated (4 °C) condition until use.
Finally an experimental diet having 40% crude protein was
prepared by mixing different feed ingredients (Table 1). The
feed was offered at the rate of 3% of wet body weight of the
fish twice a day in feeding aquaria.
Table 1: Preparation of black cumin incorporated experimental diets for growth
Ingredients
Experimental diets (% inclusion)
T1
T2
T3
T4
T5
T6
Fish Meal†
29.31
29.31
29.31
29.31
29.31
29.31
Soya meal‡
14.52
14.52
14.52
14.52
14.52
14.52
Groundnut oil cake†
9.17
9.17
9.17
9.17
9.17
9.17
Rice Powder†
14.16
14.16
14.16
14.16
14.16
14.16
Wheat Flour†
14.43
14.43
14.43
14.43
14.43
14.43
Corn flour†
11.37
11.37
11.37
11.37
11.37
11.37
Sunflower oil†
3
3
3
3
3
3
Cod liver oil†
2
2
2
2
2
2
Vitamin & Mineral Premix§
1
1
1
1
1
1
N. sativa Seeds
0.01
0.01
0.01
0.01
0.01
0.01
Betain Hydrochloride††
0.02
0.02
0.02
0.02
0.02
0.02
BHT(Butylatedhydroxytoluene)††
0.02
0.02
0.02
0.02
0.02
0.02
CMC (Carboxymethylcellulose) ††
0.99
0.99
0.99
0.99
0.99
0.99
Total
100
100
100
100
100
100
†Ingredients like fish meal, soya meal, groundnut oil cake, rice powder, wheat flour, corn flour, sunflower oil and Cod Liver Oil were procured
from local market of Kathmandu Valley.
‡Ruchi Soya Industries, Raigad, India.
§Composition of vitamin mineral mix (EMIX PLUS) (quantity 2.5kg -1)
Vitamin A 55,00,000 IU; Vitamin D3 11,00,000 IU; Vitamin B2 2,000 mg; Vitamin E 750 mg; Vitamin K 1,000 mg; Vitamin B6 1,000 mg;
Vitamin B12 6 μg; Calcium Pantothenate 2,500 mg; Nicotinamide 10 g; Choline Chloride 150 g; Mn 27,000 mg; I 1,000 mg; Fe 7,500 mg; Zn
5,000 mg; Cu 2,000 mg; Co 450 mg; Ca 500 g; P 300g; L- lysine 10 g; DL-Methionine 10 g; Selenium 50 mgl-1; Selenium 50 mgl-1; Satwari
250 mgl-1; (Lactobacillus 120 million units and Yeast Culture 3000 crore units).
†Black cuminwere obtained locally and then extracts were prepared.
††Himedia Laboratories, Mumbai, India.
2.2 Experimental design and feeding trial
A 12 week study was conducted to investigate the effects of
different water temperature on length gain, feed conversion
ratio and protein profile in brain of Indian major carp rohu
(Labeo rohita Hamilton 1822) in the wet laboratory of
Fisheries Development and Training Center, Janakpurdham,
Nepal. The experiment was conducted during
October/November and continued up to mid February so
natural climatic condition at that time was around 17-18 °C
hence 18 °C was fixed in T1 tank in the beginning of feeding
trial and after a month it was changed to 19 °C followed after
a month by 20 °C and then this tank was considered as control
having T1 (18-20 °C). Similar temperature variations were
applied in other treated tanks in which after a month in each
tank 1°C temperature was increased in each aquarium using
aquarium heater manually. Thus, experimental rearing system
consisted of18 (three replicates) glass aquaria (14”x12”x30”)
for six treatments T1 (18-20 °C), T2 (20-22 °C), T3 (22-24°C),
T4 (24-26 °C), T5 (26-28 °C) and T6 (28-30 °C) for this
experiment and in each aquarium temperature of water were
fixed with the help of aquarium heater. Four hundred fifty
fingerlings of Labeo rohita (7.25±0.27cm) were randomly
distributed in six treatment groups in triplicates following a
completely randomized design (CRD). pH ranged from 7.53
to 7.92 throughout the study. DO was maintained above 5
gm/l with the help of aerators. A randomly 5 fingerlings were
weighed randomly from each aquarium on every 15 days
interval to adjust the feeding status of carp.
2.3 Examination Procedures
2.3.1 Length gain and FCR profiles
Before harvesting, fingerlings were fasted for 24 hours and
then final length of each and individual carp were measured
for growth profiles. To understand the feed conversion ratio
(FCR) standard formula were used as FCR=F/(Wf-Wo);
Where F is the weight of food supplied to fish during the
experimental period; Wo is the live weight of fish at the
beginning of the experimental period; Wf is the live weight of
fish at the end of the experimental period.
2.3.2 Brain tissue collection for protein estimation
At the end of the feeding trial, three fish from each of the
control and experimental groups were anaesthetized with
tricaine methane sulfonate (MS-222) (5 mg l-1) for 2-3
minutes. Brain tissues were collected through surgical
dissection. The tissues samples were homogenized and then
transferred immediately to eppendorf tubes, allowed to settle
for a while then centrifuged for 5 min at 3000×g.Thus
collected brain tissues were stored at -20 °C for further
analysis. Total protein content was determined by Biuret
method developed by Doumas [11] using a kit where as
albumin was determined by BCG (Bromo Cresol Green)
method developed by Doumas [11]. Globulin was calculated by
the deduction of albumin from total protein while albumin -
globulin ratio was calculated by dividing albumin values by
globulin values.
2.3.3 Statistical Analysis
Value for each parameter measured has been expressed as
mean ± standard error of mean. The results were analyzed by
one-way Analysis of Variance (ANOVA) followed by
Duncan’s Multiple Range Test. Significance was tested at
P<0.05 level.
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International Journal of Fisheries and Aquatic Studies
3. Results and Discussion
3.1 Survival and growth performances
Survival rate and growth of a fish depends upon the
temperature and other environmental factors of aquatic
environment where fish live. In the present study fingerlings
of Labeo rohita were selected and treated with varied
temperatures in which Cent per cent survival were observed
(Figure 4 and Table 2) in fish group treated with water
temperature T4 (24-26 °C) while in other treated groups were
99.6, 98.4, 97.3, 93.7 and 93.1%, respectively. To study the
survival of fingerlings Labeo rohita was selected by Ali and
Salim [12], Saeed et al. [13] and Abid and Ahmed [14] for their
research work and found similar results. Fish requires diet as
a fuel for physical activities, body maintenance and excess of
intake diet is utilized for fish growth and sexual maturation.
Fish farmer desires maximum growth of fish by development
of muscle tissue for their economic benefit. Fish growth rate
depends upon a number of factors like species, age, health,
genetic potential, food and physicochemical properties of the
water. Better growth rate occurs in fry and fingerling as
compared to latter stage of fish because these do not show
sexual maturation. Fingerling stage offish has all the
differential organs already developed so energy from feed is
directly utilized for fish growth after maintaining all the
physiological activities.
Fig 4: Survival rate performance of Labeo rohita fed Kalaunji
(Nigella sativa) and treated by different temperature ranges.
Fig 5: Final length gain (cm) of Labeo rohita fed Kalaunji (Nigella
sativa) and treated by different temperature ranges.
Fig 6: Length gain (%) of Labeo rohita fed Kalaunji (Nigella sativa)
and treated by different temperature ranges.
Fingerlings are commonly cultured by fish farmers due to
high growth rate and high survival rate which results in to
more profits. Labeo rohita attained significantly higher body
length (10.59± 0.06 cm) and length gain % (151.28±0.68)
under water temperature range of 24-26 °C fed in fish group
of T4 aquaria (Figure 5, 6 and Table 2). Feeding management
plays an important role in fish farming for making it efficient
and profitable for fish farmers. Feeding management was
scientifically monitored throughout the study periods. The
highest length gain was observed in the fish maintained on
24-26 °C. Houlihan et al. [15], Britz et al. [16] and Azevedo et
al. [17] observed that fish were markedly influenced by the
temperature of water in which they lived. Increased length has
also been reported in Labeo rohita reared in polyhouse at
average temperature of 19 °C as compared with those in
outdoor tanks at average temperature of 14.8 °C [18]. he fish
reared in water having temperature between 24-26 °C showed
the best FCR value (1.18±0.13). The best FCR was observed
in the fish kept at 24-26 °C temperature range group by those
maintained at 22-24 and 20-22 °C. Similar results were
recorded by Andrews and Stickney (1972), who reported that
channel catfish, Ictalurus Punctatus, fingerlings reared at a
temperature range of 18-34 °C registered improvement in
FCR, with the best values obtained at 30 °C. Similar results
were also recorded by Osborne and Riddle [19].
The food consumption and wet weight production play an
important role in the increase or decrease of FCR. The FCR
values of the present study are comparable to the findings of
Rahman et al., [20]; Tareque et al., 2009 for other common
carp species but not satisfactory for Indian major carps (Singh
et al. [21]; Pramanik et al., [22] and grass carp (Dabrowski) [23],
need further study.
Goolish and Adelman [24] observed that fish reared in
increased temperature resulted in better utilization of feed in
fish than those kept under lower temperature (20.9-24.3 °C).
In contrast to the better efficiency of feed utilization at higher
temperature range, Alanara [25] did not observe any difference
in the feed efficiency of rainbow trout reared at 5 or 15 °C.
This discrepancy may be due to difference in water
temperature used in these studies. Better feed conversion ratio
of the fish maintained at 24-26 °C in this study may be
attributed to the increased feed intake of the fish, which
spared more nutrients for growth of the fish after meeting the
maintenance requirements. However, the findings of Azevedo
et al. (1998) [17] revealed that water temperature had very little
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International Journal of Fisheries and Aquatic Studies
effect on feed efficiency of rainbow trout (Oncorhynchus
mykiss). Probable explanation of improved feed efficiency of
fish maintained at higher temperature might be the increased
feed intake of the fish with increase in water temperature,
which resulted in better growth of the fish, leading to better
feed conversion ratio. Another probable explanation may be
the less energy required for the process of thermoregulation to
the fish kept at this temperature, Fig 1-8
Fig 7: Feed conversion ratio (FCR) of Labeo rohita fed Kalaunji (Nigella sativa) and treated by different temperature ranges.
Table 2: Protein profile present in different tissues of Labeorohita treated with varied temperature (°C)
Descriptive
T1
(18-20 °C)
T2
(20-22 °C)
T3
(22-24 °C)
T4
(24-26 °C)
T5
(26-28 °C)
T6
(28-30 °C)
Total Length Gain (TLG)
5.21±0.14
6.17±0.11
8.39±0.26
10.59±0.06
6.40±0.08
6.36±0.05
Length Gain (TLG %)
74.70±2.79
81.12±1.76
117.08±6.57
151.28±0.68
89.05±1.00
84.57±0.98
Feed Conversion Ratio (FCR)
2.91±0.00
2.08±0.00
1.55±0.00
1.18±0.00
1.25±0.00
1.26±0.00
Survival Rate (%)
93.74±1.21
93.16±1.13
97.31±0.15
100.00±0.00
98.48±132
99.56±0.31
Brain
protein
5.70±0.87
8.07±1.09
9.69±0.35
15.98±0.41
13.62±0.47
11.54±0.37
albumin
2.55±0.49
3.85±0.44
4.46±0.18
7.27±0.15
6.27±0.08
5.16±0.31
globulin
3.15±0.38
4.22±0.71
5.23±0.26
8.71±0.35
7.34±0.52
6.38±0.06
AG Ratio
0.79±0.08
0.94±0.12
0.85±0.05
0.84±0.03
0.86±0.07
0.81±0.04
3.2 Protein profile in brain of carp
Protein is the major dietary nutrient affecting performance of
fish [26]. It provides the essential and nonessential amino acids
which are necessary for muscle formation and enzymatic
function and in part provides energy for maintenance [27].
Several experiments have been conducted to determine the
optimal level of protein for various fish species (Ogino and
Saito, [27]; Dabrowski, [28]; Hepher, [29]; De Silva and
Anderson, [30]; Pramanik el al., [31]. The total protein, albumin,
globulin and ratio of albumin and globulin in brain of Labeo
rohita found significantly (P<0.05) higher in the group of
carp treated with temperatureT4 (24-26 °C) (Table 2).
Increase in the total serum protein, albumin and globulin
levels in brain are strongly correlated with the boost of non-
specific defense system in fishes. The A/G ratio is a
measurable humoral component of innate immune system.
Fig 8: Total protein in brain (g) of Labeo rohita fed Kalaunji (Nigella sativa) and treated by different temperature ranges.
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International Journal of Fisheries and Aquatic Studies
Fig 9: Albumin in brain (g) of Labeo rohita fed Kalaunji (Nigella
sativa) and treated by different temperature ranges.
Fig 10: Globulin in brain (g) of Labeo rohita fed Kalaunji (Nigella
sativa) and treated by different temperature ranges
Fig 11: Albumin and globulin ratio in brain (g) of carp fed Kalaunji
(Nigella sativa) and treated by different temperature ranges.
In all animals, there is a continual cycle of synthesis and
breakdown of protein with growth occurring under conditions
where the rate of protein synthesis exceeds protein breakdown
[32, 33]. In fish, the effects of various abiotic and nutritional
influences on protein synthesis and protein growth have been
studied for over 20 years and have been the subject of several
reviews [34-37]. Water temperature has been identified as the
major abiotic factor affecting the physiology and growth of
fish [38, 39]. However, although the effects of water temperature
on rates of protein synthesis in fish have been well studied
(reviewed by Haschemeyer [40]; McCarthy and Houlihan) [41],
there are very few examples where rates of protein synthesis
and growth have both been measured for the same animals [42-
45].
Our knowledge of the temperature response of protein
synthesis and protein growth in fish is still limited in a
number of respects. Any fish species exhibits thermal
tolerance over a range of water temperatures where feeding
and growth will occur and where growth performance (in
terms of both rate and efficiency) can vary according to the
water temperature [46]. However, previous studies have reared
groups of fish at a limited number of water temperatures
within the thermal tolerance range for that species, 2 °C (Reid
et al. 1995, 1997) to 10 °C [47, 48]
4. Conclusion and Recommendation
In the present experiment, the length gain percentages and
feed conversion ratio (FCR) was found comparatively better
in 24-26 °C than that of other treated groups. Protein
concentrations in brain also indicated better results in T4
treated group. Thus, it has been concluded that water
temperature ranging from 24-26 °C seemed to be the most
effective for rearing of Labeo rohita proved for better growth
and healthy carp. Aquafarmers can be benefitted by using this
technique.
5. Acknowledgement
The authors would like to thank to Dr. Narayan Giri, Chief of
Fisheries Development and Training Centre, Janakpurdham,
Nepal and the other staff members for their help during the
experiment. The authors also like to thank to the Campus
Chief Mr. Umakant Lal Karna of Amrit Campus to provide
laboratory facilities for physiological studies of brain tissue of
Labeo rohita. The author also likes to thank the research
faculty members of Integral University, Lucknow to grant the
permission for the publication of this research article by
providing MCN No. IU/R&D/2019-MCN 000588.
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