Int. J. Aquat. Biol. (2020) 8(3): 194-208
ISSN: 2322-5270; P-ISSN: 2383-0956
Journal homepage: www.ij-aquaticbiology.com
© 2020 Iranian Society of Ichthyology
Original Article
Evaluation of probiotic adequacy, immunomodulatory effects and dosage application of
Bacillus coagulans in formulated feeds for Catla catla (Hamilton 1822)
Anita Bhatnagar*,1Shashi Raparia
Department of Zoology, Kurukshetra University, Kurukshetra -136119, India.
s
Article history:
Received 19 January2020
Accepted 2 June 2020
Available online 2 5 June 2020
Keywords:
Hydrophobicity
Probiotic properties
Phagocytic ratio
Indian carp
Abstract: The present study was conducted to study the probiotic properties, antagonistic effect
against pathogenic Aeromonas hydrophila of Bacillus coagulans isolated from intestine of
healthy Catla catla Hamilton, 1822; and its optimum dosage for growth promotion and
immunostimulation. The isolated B. coagulans from the gastrointestinal tract of C. catla was first
assessed for its probiotic properties viz., antagonism towards pathogen and cell surface adhesion. A
feeding trial of 90 days was conducted to optimize the inclusion level of B. coagulans in diets
and C. catla fingerlings (avg. wt. 0.30±0.03g) were fed on feed supplemented with 1x103 (diet D1),
2x103 (diet D2), 3x103 (diet D3) and 5x103 (diet D4) B. coagulans CFU g-1 of feed in triplicate
treatments. The growth and digestibility parameters, intestinal enzyme activities were significantly
higher in group of fish fed on feed D3 (3x103 CFU g-1) in comparison to other dietary treatments
except for food conversion ratio which was significantly higher in control group. Significantly higher
value of carcass protein level, lower excretion of metabolites (ammonia and phosphates),
enhancement of non-specific immune response and increase of total Erythrocyte count (TEC) and
total Leucocyte Count (TLC) were observed in fish fed with probiotics supplemented diets. The
results obtained in the present study support the use of B. coagulans for better growth and proper
nutrient utilization. The broken line analysis was carried out and polynomial fit curve further suggest
that the optimum concentrations of B. coagulans as high as 3000 (3x103) CFU g-1 of feed is required
for improving the overall physiological performance and enhancement of defense mechanisms in the
fingerlings of C. catla.
Introduction
Aquaculture is most promising, viable and fast-
growing sector to provide nutritional security and its
intensification is required to keep pace with surging
need of animal protein. Intensification increases stress
level in the animal as well as the environment. Disease
outbreak is considered as most important constraint to
its continued expansion. The application of antibiotics
and chemotherapeutics to control diseases has led to
serious problems such as the evolution of drug
resistant pathogens, suppression of the aquatic
animal's immune system, significant risk to human
health and environmental hazards (Brogden et al.,
2014; Allameh et al., 2015). An alternate approach to
enhance disease resistance, immune responses and
other health benefits is the administration of probiotics
*Correspondence: Anita Bhatnagar
E-mail: anitabhatnagar@gmail.com
which play an important role in improving health of
fish (Bandyopadhyay et al., 2015; Sivagami and
Ronald, 2018). Merrifield et al. (2010) defined
probiotics “as any microbial cell provided via the diet
or rearing water that benefits the host fish, fish farmer
or fish consumer, which is achieved, in part at least,
by improving the microbial balance of the fish”.
Many studies have reported that probiotic
supplemented diets have a major impact on growth
performance of fish (Gao et al., 2016; Bhatnagar and
Lamba, 2017; Liu et al., 2018; Gobi et al., 2018;
Sivagami and Ronald, 2018; Ullah et al., 2018).
Probiotic are also known to improve intestinal
enzymatic activities in fishes (Sivani et al., 2016;
Makled et al., 2019). Zhang et al. (2018) reported that
supplementation of Lactobacillus delbrueckii as
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Int. J. Aquat. Biol. (2020) 8(3): 194-208
probiotic enhanced growth performance and intestinal
enzymatic activities as well as whole body
composition of common carp. Selection of probiotics
demands that is should be isolated from the
gastrointestinal tract of host species intended to study
(Patel et al., 2010; Makled et al., 2019) as
commercially available probiotics are mainly from
non-fish sources, which are believed to be unable to
survive and/or remain viable at optimum
concentrations in the fish intestine (Ghosh et al.,
2008). Probiotics isolated from mature animals are
well accepted in feeds of immature animals of same
species (Gomez-Gil et al., 2000; Ghosh et al., 2003).
Bacillus has been evaluated as probiotics in fish due
to its antagonistic property, ability to enhance growth
and immune response and is environment friendly to
use (Shelby et al., 2006; Sumathi et al., 2014;
Bhatnagar and Lamba, 2015, 2017; Bhatnagar and
Saluja, 2019; Bhatnagar and Dhillon, 2019; Bhatnagar
and Rathi, 2020). Bacillus circulans and Bacillus sp.
have been isolated from the gut of Catla catla and
Cirrihinus mrigala and their effect on growth,
nutritional quality and immunity have been studied
when incorporated in formulated diets
(Bandyopadhyay and Patra, 2004). Studies were
undertaken to isolate gut adherent potential probiotic
bacterium to improve fish growth and digestibility in
C. catla (Bhatnagar et al., 2012; Bhatnagar and
Raparia, 2014; Bhatnagar and Saluja, 2019), and it
was observed that significantly high growth
performance can be achieved in the group of fishes fed
on diet containing B. coagulans. In our earlier studies,
three doses 200, 2000 and 20000 CFU per gram were
used (Bhatnagar and Raparia, 2014), and it was
found that optimum dose is somewhere near 2000
CFU g-1, therefore to evaluate the proper dose four
doses 1000, 2000, 3000 and 5000 CFU g-1 were used
in the present study. However, it was felt that there is
a need to assess the probiotic properties of this
bacterium and its impact on growth performance,
nutritional physiology and hematological parameters.
Therefore, the present studies were conducted to
evaluate the antagonistic properties of this probiotic
species against Aeromonas hydrophila (major disease-
causing agent in water), its influence on growth
performance, blood parameters and immunity of
C. catla and its optimum inclusion level in formulated
diets.
Materials and Methods
Experimental Animals: Fingerlings of C. catla were
obtained from “Sultan Fish Seed Farm” in Butana,
Nilokheri, District Karnal (Haryana, India).
Fingerlings were kept in glass aquarium of 30L
capacity in laboratory conditions where temperature
was maintained at 25±1°C. Fishes were acclimatized
for 10 days prior to experiment start. Each aquarium
was filled with de-chlorinated tap water and then
stocked with 20 fingerlings with average body weight
(BW) 0.30±0.03g. During the experiment, the water
samples from all the aquaria were collected fortnightly
and temperature, dissolved oxygen (DO), pH,
electrical conductivity, calcium, chlorides and total
alkalinity were measured following American Public
Health Association (2017) to investigate the influence
of supplemented feeds on quality of holding water. At
the end of feeding trials, water samples from each tub
were collected at two-hour intervals for the estimation
of excretory levels of total ammonia (N-NH4+) and
reactive orthophosphate following APHA (2017), and
calculated following Sumagaysay-Chavoso (2003).
Mass culture of B. coagulans and feed preparation:
Bacillus coagulans, isolated from gut of C. catla
(Bhatnagar et al., 2012) was used in the present
studies. It was kept in nutrient agar slant at 4oC for
further use. Bacillus coagulans was inoculated into
conical flask (500 ml) containing nutrient broth and
incubated at 30oC for 24 h in shaker incubator. The
culture was centrifuged at 10000 rpm for 20 minutes
at 4°C and supernatant was discarded, while the
pellets were resuspended in phosphate buffer saline
(PBS; pH 7.2). The suspension was similarly washed
and recentrifuged four times and then quantified by
spread plate technique (nutrient agar), incubated at
30oC for 24 h to determine the number of colonies
forming units (CFU) (Bhatnagar and Raparia, 2014).
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Bhatnagar and Raparia / Bacillus coagulans as probiotic bacterium and its dosage for Catla
Feed was prepared by thoroughly mixing the
ingredients (Table 1) followed by steaming for 20
minutes, cooled mixed thoroughly and then pellets
were made by a hand pelletizer. Feed were air dried,
probiotic were sprayed in respective concentrations
and finally stored in vacuum sealed plastic containers
at 4°C. In all the treatments, fishes were fed with
respective diets daily at 4% BW in two instalments at
8:00 and 16:30 hours for 90 days. Growth parameters
and enzymatic analysis was done using standard
methods. Biochemical analysis of feed and fish
carcass was carried out following AOAC (2019).
Antibiotic resistance study: Bacillus coagulans was
examined for its inhibitory effects against the
pathogenic A. hydrophila (IMTECH, Chandigarh)
using Well diffusion assay (Lyon and Glatz, 1993).
Aeromonas hydrophila was spread by a sterile swab,
evenly, over the face of a sterile agar plate. A
microbial suspension of each intestinal bacterial strain
was applied in well at the centre of the agar plate (in a
fashion such that the antimicrobial doesn't spread out
from the centre) and incubated for 24 hours at 30º C to
check the prevention of Aeromonas growth by the
antibiotic activity. The strains that showed halos larger
than 20 mm were considered positive.
Hydrophobicity assay: The cell surface
hydrophobicity was evaluated according to the ability
of the microorganisms to partition into hydrocarbon
from phosphate buffer solution using the method of
Savage (1992). Bacterial isolates were grown in
nutrient broth (Merck, Germany) at 37°C for 24 h.
After being centrifuged at 5000 rpm for 15 min, the
pellets (bacterial precipitates) were washed twice with
phosphate buffer solution and optical density (OD450)
of the bacteria at 450 nm adjusted to 0.5 A. About1 ml
of bacterial suspension was added with 60 μl of a
hydrocarbon viz., xylene (Fisher, England) and
toluene (Merck, Germany), and vortexed for 1 min
followed by determination of optical density of the
water phase. Hydrophobicity was calculated
according to the equation: [(OD450 before – OD450
after)/OD450 before] ×100 = % hydrophobicity.
Growth experiment
Experimental setup: The experiment was conducted
Table 1. Ingredient and Proximate composition (% dry weight basis) of experimental diets.
Dietary treatments
DC
(control)
D1
(1000 CFU g-1)
D2
(2000 CFU g-1)
D3
(3000 CFU g-1)
D4
(5000 CFU g-1)
Ingredient composition
Groundnut oil cake 650.0 650.0 650.0 650.0 650.0
Rice bran 42.0 42.0 42.0 42.0 42.0
Processed soybean* 266.0 266.0 266.0 266.0 266.0
Wheat flour 32.0 32.0 32.0 32.0 32.0
Chromic oxide (Cr2O3) 10.0 10.0 10.0 10.0 10.0
Mineral mixture** 10.0 10.0 10.0 10.0 10.0
Probiotic bacterium (cells g-1)
Bacillus coagulans
0.0 1000 2000 3000 5000
*Soybean was hydrothermally processed in an autoclave at 121°C (15 lbs for 15 minutes) to eliminate antinutrient factors
(Garg et al., 2002). **Each kg has nutritional value: copper 312 mg, cobalt 35 mg, magnesium 2.114g, iron 979 mg, zinc
2 mg, iodine 15 mg, DL-methionine 1.920 g, L-lysine monohydrochloride 4.4 g, calcium 30%, phosphorous 8.25%.
Proximate composition
Crude protein (%) 39.80±1.36 A 38.24±0.98A 39.51±0.86 A 39.86±0.79 A 39.35±0.79 A
Crude fat (%) 9.10±0.26 B 9.21±0.24 B 9.7±0.31 A 9.29±1.26 B 9.18±1.26 B
Crude fiber (%) 6.23±0.06 A 6.13±0.07 A 6.28±0.08 A 6.37±0.06 A 6.14±0.06 A
Total ash (%) 6.64±0.39 A 6.66±0.34 A 6.51±0.26 A 6.42±0.47 A 6.45±0.47 A
Moisture (%) 7.41±0.20 A 7.37±0.28 A 7.39±0.19 A 7.22±0.37 A 7.38±0.37 A
Nitrogen free extract (%) 30.82±1.42 B 32.38±1.49 A 31.84±1.11 AB 30.8±1.07 AB 31.5±1.07 AB
Gross energy (kJ g-1) 17.90±0.09 B 18.53±0.18 A 18.63±0.08 A 18.37±0.09 A 18.33±0.09 A
Feed phosphorus (%) 1.48±0.11 C 1.42±0.08 C 1.54±0.20 B 1.62±0.07 A 1.47±0.07 C
All values are Mean±S.E of mean. Means with different letters in the same row are significantly (P<0.05) different. (Duncan’s Multiple
Range test).
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under laboratory conditions (25±1°C) in glass
aquarium (30 L capacity) at Aqaculture Research Unit
of Department of Zoology, Kurukshetra University,
Kurukshetra. Each aquarium was filled with de-
chlorinated tap water and then stocked with 20 fish
fingerlings with average BW 0.3±0.03 g.
Five dietary treatments (DC, D1, D2, D3 and D4) were
performed with three replicates of each treatment. In
treatment 1 (DC), fishes were fed on artificial diet
without probiotic bacteria (i.e., control diet).
Ingredient composition in g kg-1: ground nut oil cake,
650; rice bran 42; hydrothermically processed
soybean 266; wheat flour 32; mineral mixture 10. In
treatment 2 (D1), fishes were fed on diet containing
B. coagulans suspension in proportion 1000 (1x103)
CFU g-1 of feed. In treatment 3 (D2), fishes were fed
on artificial diet containing B. coagulans in proportion
of 2000 (2x103) CFU g-1 of feed. In treatment 4 (D3),
fishes were fed on artificial diet containing B.
coagulans in proportion of 3000 (3x103) CFU g-1 of
feed and in treatment 5 (D4), with proportion of 5000
(5x103) CFU g-1 of feed. All these diets were
isocaloric and isoproteic with approximately 40%
protein content. After spraying, the feed was air dried
at room temperature and the bacterial concentration of
feed (CFUg-1) was calculated. Finally, the feed was
stored in vacuumed plastic container at 4oC. All
groups of fish were fed daily at 4% BW in 2
installments at 08:00 and 16:30 hours for 90 days.
Growth parameters and enzymatic analysis was done
using standard methods. Biochemical analysis of feed
and fish carcass was carried out following AOAC
(2019).
Blood parameters study: After the end of feeding trial,
blood samples were collected from fingerlings of each
dietary treatment for hematological diagnosis by using
a heparinized syringe from caudal vein or heart by
cardiac puncture (Lavanya et al., 2011). Blood
samples of five fishes were pooled for analysis. EDTA
(1 mg EDTA ml-1) was used as anticoagulant in blood.
Total Erythrocyte count (TEC) and total Leucocyte
Count (TLC) were estimated by analyzing the blood
samples from each treatment with the help of
hemocytometer using a Neubauer’s counting chamber.
Nonspecific immune response: Blood samples were
heparinsed and immediately used for phagocytic assay
(Park and Jeong, 1996).
Phagocytic assay: For phagocytic assay cells of
freshly grown pathogenic bacteria A. hydrophila in
0.1 ml of PBS were added to 0.1 ml of blood sample
(pooled samples of blood of five fishes mixed with
EDTA as anticoagulant) of fishes of each treatment in
sterile microplate. Blood was then incubated for 30
min at 25°C after thorough mixing in the well. The
plate was removed and fifty ml of each suspension was
transferred on glass slides to make smears. After air
drying, smear was fixed in 95% ethanol, re-dried and
stained with May Grunwald Giemsa. The phagocytic
cells and phagocytosed bacteria were enumerated.
Phagocytic ratio (PR) and phagocytic index (PI) were
determined by enumerating 100 phagocytes per slide
under a microscope. The average of three slides was
calculated depending on the formula which is given
below.
Phagocytic ratio (PR; i.e. percentage of cell with
engulfed bacteria) = (No. of phagocytic cells with
engulfed bacteria/No. of phagocytic cells) × 100.
Phagocytic index (PI; i.e. number of engulfed bacteria
per cell) = No. of engulfed bacteria/No. of phagocytic
cells.
Nitroblue tetrazolium (NBT) assay: The oxygen
radical production by blood phagocytes during
respiratory burst activity was measured through
nitroblue tetrazolium (NBT) assay as described by
Anderson and Siwicki (1995). Briefly, 0.1 ml of
EDTA mixed blood from each treatment group was
taken in Eppendorf to which 0.1 ml of 0.2% NBT
solution was added. The mixture was incubated for 30
minutes at 25°C. From the suspension, 50 µl was
taken, added to 1.0 ml N, N-dimethyl formamide in a
glass tube and centrifuged at 3000g for 5 minutes. The
optical density (OD) of the supernatant was measured
at 540 nm in the spectrophotometer.
Challenge trial: After feeding for 90 days, 10 fishes
from each treatment were challenged with
A. hydrohila which has been cultured and maintained
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Bhatnagar and Raparia / Bacillus coagulans as probiotic bacterium and its dosage for Catla
in the selective medium. Fishes from each replicate
were immersed in a suspension of A. hydrophila ~ 105
CFU ml-1 followed by a second immersion ~107 CFU
ml-1 after 7 days (Austin et al., 1995). Per cent survival
was measured for 10 days based on observation that
mortality reached its plateau after one week (Sahoo et
al., 1998) and relative percentage survival was
calculated by the following formula (Ellis, 1998)
below:
RPS = 1- (Percent mortality in treated group/ Percent
mortality in control group) × 100
Statistical analysis: Data were examined by one-way
analysis of variance (ANOVA). When ANOVA
identified differences among groups, a multiple
comparison, Duncan’s test was conducted to examine
significant differences among treatments using
Statistical Package for Social Science (SPSS-11.5)
and significant differences were declared at P≤0.05.
Data of experiments were further subjected to
orthogonal polymonials (broken line regression
analysis) for trend analysis (Zeitoum et al., 1976).
Results
Antagonistic activity of B. coagulans/antibiotic
resistance assay: The size of zone of inhibition was
found to be 19.0±1.6 and ranged between 15 to 21 mm
(Fig. 1).
Hydrophobicity assay: The use of xylene, and toluene
to evaluate the hydrophobic cell surface properties of
the tested B. coagulans showed consistent positive
results. The hydrophobicity of B. coagulans was
30.49±0.84% in xylene, and 22.79±3.96% in toluene.
Proximate composition: The average proximate
composition of formulated diet revealed that the diets
were isonitrogenous. Values of moisture, crude
protein, crude fat, total ash, crude fiber and NFE are
shown in Table 1 as % dry weight basis.
Fish growth, survival, digestibility and nutrient
retention: The growth responses of test fish fed on
experimental diets (DC to D4) are shown in Table 2.
Fish satisfactorily accepted the experimental diet from
the beginning of the experiment and maintained
normal behavior throughout the experimental period.
Survival rate (%) was high in all dietary treatments
and slight mortality occurred only during the initial
days of experiment. Statistical analysis revealed that
the growth of fish in terms of weight gain (g), growth
per cent gain (%) in body weight, growth per day (%)
in BW and specific growth rate (SGR) were
significantly (P<0.05) higher in treatment D3 in
comparison to dietary treatments DC, D1, D2 and D4.
Also, significantly (P<0.05) higher values of
digestibility parameters viz., apparent protein
digestibility (APD), gross conversion efficiency
Table 2. Growth performances and intestinal enzyme activities of Catla catla fed on Soybean based diets containing varying proportions of
probiotic bacterium Bacillus coagulans.
Growth parameters
Dietary treatments
DC
(Control)
D1
(1000CFUg-1)
D2
(2000CFUg-1)
D3
(3000CFUg-1)
D4
(5000CFUg-1)
Initial length (cm) 1.95±0.05A 2.0±0.04A 1.90±0.06A 1.95±0.04A 2.05±0.02A
Initial weight (g) 0.32±0.02A 0.29±0.01A 0.31±0.02A 0.32±0.03A 0.31±0.02A
Final weight (g) 1.40 ±0.03E 1.87±0.04C 2.08±0.05B 2.30±0.03A 1.69±0.02D
Live weight gain (g) 1.09±0.02E 1.57±0.04C 1.77±0.04B 1.98±0.03A 1.4±0.02D
Survival rate (%) 100A 100A 100 A 100 A 98.3±1.36A
Growth (%) gain in BW 349.3±17.09E 524.3±28.4C 572.7±17.7B 635.6±28.5A 445.9±31.5D
Growth/day (%) in BW 1.35±0.03C 1.56±0.03B 1.63±0.02AB 1.77±0.02A 1.5±0.04 BC
Specific growth rate (SGR) (%BW d-1) 0.72±0.02C 0.85±0.02AB 0.88±0.02A B 0.99±0.01A 0.81±.03B
Feed conversion ratio (FCR) 2.6±0.09 A 1.96±0.04B 1.74±0.02C 1.56±0.03D 1.99±0.04B
Gross conversion efficiency (GCE) 0.41±0.02D 0.53±0.01C 0.65±0.01B 0.80±0.02A 0.59±0.01C
Protein efficiency ratio (PER) 1.18±0.05E 1.34±0.03C 1.45±0.01B 1.67±0.02A 1.2±0.03D
Apparent protein digestibility (APD)
(%)
73.3±0.64E 79.8±0.42C 81.5±0.57B 86.4±0.46A 77.7±0.72D
All values are Mean±S.E of mean. Means with different letters in the same row are significantly (P<0.05) different. (Duncan’s Multiple
Range test)
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(GCE) and protein efficiency ratio (PER) and
significantly (P<0.05) lower FCR (1.56±0.03) were
observed in the dietary treatment D3. The highest FCR
was found in the control group (2.6±0.09) (Table 2).
The data on weight gain revealed that initially up to 15
days not much variations were observed in the weight
gain of group of fishes fed on varying dietary
treatments. However, growth rate increased
significantly (P<0.05) in the fishes fed on diet D3 after
30 till 90 days (Fig. 2). Diet containing B. coagulans
at 3000 CFU g-1 of diet depicted 81.65% increase in
weight gain in comparison to control diet (Fig. 3).
Data of experiments were further subjected to
orthogonal polymonials (broken line regression
analysis) for trend analysis, also showed a clear dose
dependent trend line curve. Polynomial curve fitting
to the data of weight gain in the fingerlings of C. catla
is shown in Figure 4.
Intestinal digestive enzyme activities: Intestinal
digestive enzyme activities for protease, amylase and
Table 3. Proximate carcass composition of Catla catla fed on Soybean based diets containing varying proportions of probiotics bacterium Bacillus
coagulans.
Proximate composition Initial value
Dietary treatments
DC
(control)
D1
(1000 CFUg-1)
D2
(2000 CFUg-1)
D3
(3000 CFUg-1)
D4
(5000 CFUg-1)
Moisture (%) 73.07±0.36 70.65±0.51A 69.5±0.34AB 68.62±0.32B 66.10±0.34C 68.5±0.34B
Crude protein (%) 8.90±0.06 11.93±0.21D 14.41±0.07C 16.34±0.19B 17.04±0.21A 13.99±0.07C
Crude fat (%) 2.2±0.04 5.77±0.07A 3.97±.06 BC 4.45±0.15B 3.75±0.06C 4.07±0.07BC
Total ash (%) 3.6±0.06 4.27±0.18A 4.15±0.10A 3.95±0.09B 3.93±0.04B 4.29±0.10A
Nitrogen free extract (%) 12.2±0.40 7.4±0.15C 8.60±0.54B 8.51±0.45B 8.82±0.20A 8.40±0.36B
Gross energy (kJ/g) 5.06±.06 6.36±0.08D 6.3±0.05D 6.74±0.04B 7.07±0.05A 6.5±0.05C
Phosphorus (%) 0.53±0.02 0.59±0.03D 0.71±0.02AB 0.67±0.03C 0.69±0.03B 0.73±0.02A
All values are Mean±S.E of mean. Means with different letters in the same row are significantly (P<0.05) different. (Duncan’s
Multiple Range test).
Figure 1. Antagonistic activity shown by probiotic
bacterium Bacillus coagulans for pathogenic
Aeromonas hydrophila.
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Bhatnagar and Raparia / Bacillus coagulans as probiotic bacterium and its dosage for Catla
cellulase were determined. It was found that specific
activity of digestive enzymes was significantly
(P<0.05) higher in all the dietary treatments in
comparison to control group. The values showed an
increasing trend from treatment DC to D3. Thereafter,
with further increase in the inclusion level of probiotic
bacteria (Diet-D4), containing B. coagulans in
proportion of 5000 CFU g-1 of feed, the values
decreased (Fig. 5).
Fish carcass composition: Initial and final carcass
composition with respect to proximate nutrients of test
fish on the basis of feeding trial is shown in Table 3.
Crude protein (%) and gross energy (kJg-1) were found
to be significantly (P<0.05) higher in the carcass of
fish fed on diet D3. Moisture (%) and crude fat (%)
Figure 2. Increase in mean fish weight (g) ±S.E of mean of Catla
catla fingerlings fed on diets supplemented with varying
proportions of probiotics Bacillus coagulans (DC=control,
D1=1000 cells g-1, D2=2000 cells g-1, D3=3000 cells g-1 and
D4=5000 cells g-1 of diet) from day 15 to 90.
Figure 3. Polynomial fit curve using broken line analysis to show
effect of Bacillus coagulans supplementation (DC=control,
D1=1000 cells g-1, D2=2000 cells g-1, D3=3000 cells g-1 and
D4=5000 cells g-1 of diet) fitting to the data of weight gain in the
fingerlings of Catla catla.
Figure 4. Per cent increase in growth of Catla catla fed on varying
dietary treatments containing varying proportion of Bacillus
coagulans (DC=control, D1=1000 cells g-1, D2=2000 cells g-1,
D3=3000 cells g-1 and D4=5000 cells g-1 of diet).
Figure 5. Intestinal Enzyme activities of Catla catla fed on varying
dietary treatments containing varying proportion of Bacillus
coagulans (DC=control, D1=1000 cells g-1, D2=2000 cells g-1,
D3=3000 cells g-1 and D4=5000 cells g-1 of diet).
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was found to be significantly (P<0.05) higher in
dietary treatment DC. Nitrogen free extract (NFE) was
found to be higher in diet D3. However, no significant
(P<0.05) variations were observed in total ash (%) of
carcass of fishes fed on different diets.
Effect of experimental diets on water quality
characteristics: The data on water quality
characteristics pertaining to five dietary treatments is
presented in Table 4. In general, significant low values
in total ammonia excretion and reactive phosphate
production (mg Kg-1 BW d-1) were recorded in fish fed
on diet D3 supplemented with 3000 CFU g-1 of feed.
Haematological parameters: The RBC was
significantly higher (P<0.05) in fishes fed on diet D3
(2.4±0.08) than in the control treatment DC
(1.33±.03). In the present study, significant increase
(P<0.05) in WBC count was observed in fishes of
treatment D3 (50.5 ± 2.1) when compared to control
treatment DC (20.7± 0.82). Among the post-challenge
groups, DC showed significantly (P<0.05) lower RBC
than the others. The post-challenge data showed
increase in leukocyte count irrespective of the
B. coagulans inclusion signify a possible increased
infection and inflammatory response mediated by
leukocyte against bacteria (Table 5; Fig. 6).
Phagocytic responses: Phagocytic ratios and
phagocytic indices in the fish fed with varying
proportion of B. coagulans were significantly (P<.05)
higher than in control fish during the assay period. The
highest values of phagocytic ratio (79.01±1.72) and
phagocytic index (2.61±0.05) were observed in
dietary treatment D3 and the lowest in fish fed on the
control diet (59.58±1.19 and 1.75±.02, respectively).
(Table 6; Fig. 7).
Table 4. Effect of fish fed on soybean-based diets with different proportion of probiotic bacterium Bacillus coagulans supplementation on water
quality characteristics.
Physico-chemical parameters
Dietary treatments
DC
(control)
D1
(1000 CFUg-1)
D2
(2000 CFUg-1)
D3
(3000 CFUg-1)
D4
(5000 CFUg-1)
Dissolved oxygen (DO) mgL-1 6.4±0.07A 6.1±0.02C 6.4 ±0.08A 6.3±0.10A 6.1±0.10A
pH 7.80±0.01A 7.79±0.02A 7.82±0.01A 7.84±0.01A 7.78±0.01A
Conductivity (µ mho cm-1) 624.66±3.32B 629±3.42B 687.33±3.61A 685.83±2.68A 644.83±2.68AB
Alkalinity(carbonates) 21.33±0.48B 22.61±0.79AB 24.76±0.33A 24.80±0.58A 24.2±0.58B
Alkalinity(bicarbonates) 128.63±3.87C 144.54±4.17AB 149±5.33A 143.5±4.20B 144.5±4.20 AB
Chloride (mg L-1) 24.36±0.76A 20.87±0.57B 25.3±1.05A 24.98±0.87A 25.08±0.87A
Calcium (mgL-1) 25.17±1.08AB 24.19±0.96B 22.73±0.53C 26.41±0.98A 19.41±1.92D
Total dissolved solids 575.5±16.77A 539.51±8.53B 458.33±14.53E 479.30±19.27D 486.30±18.27C
Total NH3-Nexcretion (mg Kg
-1
BW day-1)
1890.2±32.74A 1287.31±19.4C 752.8±16.36D 619.3±13.4E 1326.3±19.9B
Total O-PO4 production (mg Kg
-1
BW day-1)
766.02±11.3A 472.62±7.55C 335.16±8.07D 278.55±13.1E 473.15±13.6B
Means with different letters in the same row are significantly (P<0.05) different. (Duncan’s Multiple Range test).
Table 5. Hematological Values of Catla catla fed on Soybean based diets containing varying proportions of probiotics bacterium Bacillus
coagulans.
Treatments
Haematological parameters
RBC (106mm3) WBC (103mm3)
Pre-Challenge Post Challenge Pre challenge Post Challenge
DC (Control) 1.33±0.03E 1.01±0.04 D 20.7±0.82 E 22.58±0.97D
D1 (1000 CFUg-1) 1.64±0.06C 1.51±0.03C 32.3±1.2C 36.52±1.4C
D2 (2000 CFUg-1) 1.96±0.04B 1.84±0.06B 39.3±1.6B 43.67±1.9B
D3 (3000 CFUg-1) 2.4±0.08A 2.15±0.03A 50.5±2.1A 53.58±2.5A
D4 (5000 CFUg-1) 1.44±0.04D 1.08±0.02D 25.3±0.7D 34.76±1.8C
All values are Mean±S.E of mean. Means with different letters in the same column are significantly (P<0.05) different.
(Duncan’s Multiple Range test).
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Bhatnagar and Raparia / Bacillus coagulans as probiotic bacterium and its dosage for Catla
NBT assay: Respiratory burst activity of phagocytes
was measured by reduction of Nitro Blue Tetrazolium
(NBT) by intracellular superoxide radicals produced
by leukocytes. The production of superoxide radicals
was significantly influenced by the probiotic diets.
Maximum increase in the NBT reduction value was
observed in treatment D3 (Fig. 8).
Survival rate with challenge test: After challenge
with A. hydrophila, the first mortality was recorded
after 24 h. Mortality was recorded up to 10 days after
challenge. Significantly (P<0.05) higher mortality
(73.3%) was recorded in fishes of control group. The
data on relative per cent survival is presented in the
form of survivorship curve (Fig. 9). Treatment D2 and
D3 fed groups showed significantly (P<0.05) higher
relative percent survival, 86.36 and 90.9%
respectively.
Clinical signs observed after challenge: The fish were
sluggish and gradually lost their equilibrium 24-48 h
after challenge with A. hydrophila. The clinical signs
were characterized by hyperemic condition on the
ventral side of the body, a visibly swollen abdomen
and a slightly protruding reddish vent. The eyes of the
infected fish were opaque and during the terminal
stages the animals were seen floating dorsal side down
at the water surface. The abdomen was distended due
to accumulation of fluid in the peritoneal cavity. These
changes were not evident in D3 group. Mortality
Table 6. Effect of Bacillus coagulans supplementation on phagocytic ratio and phagocytic index of Catla catla.
Peripheral blood monocytes
Fish group
Phagocytic
index
Phagocytic
ratio (%)
Bacteria Cells
within
phagocytes
No. of
ingesting
phagocytes
Total no. of
phagocytes
1.75±0.02D 59.58±1.19E 72.67±3.18 41.33±1.76 69.33±2.18 DC (Control)
2.06±0.07C 68.39±1.74C 95±2.30 46.4±2.4 67.6±2.02 D1 (1000 CFUg-1)
2.31±0.03B 73.17±1.09B 135.8±6.17 58.3±1.91 79.7±2.12 D2 (2000 CFUg-1)
2.61±0.05A 79.01±1.72A 173.6±3.67 66.3±1.83 84.2±2.41 D3 (3000 CFUg-1)
1.95±0.04C 63.5±2.41D 86±2.88 44±1.52 69.4±0.98 D4 (5000 CFUg-1)
All values are Mean±S.E of mean. Means with different letters in the same column are significantly (P<0.05) different. (Duncan’s
Multiple Range test).
Figure 6. (A) Erythrocytes (400X), (B) Thrombocytes
(a), Basophils (b), and Lymphocytes (c) and (C)
Macrophages (a) and Early phagocytic cell (b), of Catla
catla in D3 treatment after 90 days of feeding trial
(1000X).
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Int. J. Aquat. Biol. (2020) 8(3): 194-208
percentage was as low as 10.0±5.7 and 6.6±3.3 in
treatment D2 and D3, respectively.
Discussions
In the present study, the novel strain of B.coagulans
isolated from the gut of C. catla (Bhatnagar et al.,
2012) was tested for antagonistic effect on the growth
of common indicator fish pathogen A. hydrophila by
the appearance of clear inhibition zone by well
diffusion assay. The results revealed a clear zone of
inhibition ranging from 15 to 21 mm with a mean
value of 19.0±0.9 mm (Plate-6) clearly, indicating that
this strain of B. coagulans can limit the growth of fish
pathogen A. hydrophila by producing antimicrobials.
Urdaci and Pinchuk (2004), Bhatnagar and Lamba
(2015) and Bhatnagar and Dhillon (2019) have also
reported that Bacillus species could produce a large
number of antimicrobials.
The potential of probiotics is further inferred
through the ability to adhere and to colonize in the
intestinal tract. The hydrophobicity of this strain of
B. coagulans was 30.49±0.84% in xylene and
Figure 7. (A) Early Phagocytic cell, (B) Phagocytic
cells and(C) Mature Phagocytic cells, of Catla catla in
D3 treatment after 90 days of feeding trial (1000X).
Figure 8. Nitro Blue Tetrazolium (NBT) activity of Catla catla fed
on diet containing various proportions of probiotic bacterium
Bacillus coagulans.
Figure 9. Survivorship curve of Catla catla in different dietary
treatments containing varying proportion of Bacillus coagulans
(DC=control, D1=1000 cells g-1, D2=2000 cells g-1, D3=3000 cells
g-1 and D4=5000 cells g-1 of diet) challenged with Aeromonas
hydrophila.
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Bhatnagar and Raparia / Bacillus coagulans as probiotic bacterium and its dosage for Catla
22.79±3.96% in toluene, clearly revealing that this
strain can colonize the gut of C. catla and has
properties of successful probiotic. Mahdhi et al.
(2011) have also advocated the ability of B. subtilus
and B. coagulans to adhere to the intestinal surface and
reported hydrophobicity with toluene 30.3±9.40 and
31.3±3.70 and with xylene 32.2±5.60 and 36.10,
respectively. Bhatnagar and Lamba (2015) and
Bhatnagar and Dhillon (2019) have also characterized
properties of probiotics on the basis of
hydrophobicity. These findings of the present study
suggested that the isolated B. coagulans has potential
to be the probiotic bacterium for C. catla.
In the present study, attempt has also been made to
evaluate the optimum dose of probiotic
supplementation in the formulated feed for C. catla.
The optimum probiotic level which resulted in highest
growth in C. catla fingerlings in terms of live weight
gain (g), growth per cent gain, SGR (specific growth
rate) and nutrient retention (PER, GCE and APD) was
found to be around 3000 CFU g-1 of feed (treatment
D3). The polynomial fit curve (broken line regression
analysis) of weight gain also represented the optimum
dose at dietary treatment D3 (B. coagulans @ 3000
CFU g-1) with high R2 values (0.9637, y=-0.0425x3 +
0.2318x2+0.0043x+0.912). FCR (feed conversion
ratio) values decreased with each increase in the
dietary probiotic content upto 3000 CFU g-1 of feed.
Thereafter, further increase in dietary probiotic level
resulted in increase in FCR and growth depression.
The findings of the present study showed similarity
with the study of Sivani et al. (2016) in which
inclusion rate of probiotic bacterium increased after a
certain level, a decrease in growth performance was
observed. Although, all the feeds were isonitrogenous
but the concentration of probiotics in dietary treatment
D3 might have been helpful for proper nutrient
utilization. High carcass crude protein and lesser
nitrogen and phosphate excretion were also observed
in dietary treatment D3 which can be attributed to
proper probiotic concentration, whereas lesser carcass
protein and greater nitrogen and phosphate excretion
were observed in dietary treatment D4 which could
have been due to the overall low feed utilization level.
The high APD (apparent protein digestibility)
values for the diet containing B. coagulans at 3000
CFU g-1 of diet may be attributed to high dietary
utilization. Ghosh et al. (2003) using B. circulans as
probiotic in Labeo rohita fingerlings; Rengpipat et al.
(1998) using Bacillus sp. as probiotics in Paneus
monodon, Bhatnagar and Lamba (2015) using
B. cereus in C. mrigala, Bhatnagar and Dhillon (2019)
using Aneurinibacillus aneurinilyticus for L. calbasu
also reported high values of APD values at doses
coinciding with high growth performance.
The enhanced enzyme activity level in the gut
because of extracellular enzyme production by
B. coagulans might have helped in increasing the food
absorption and thus resulted in high growth in
treatment D3. Rani et al. (2004), Bhatnagar and
Khandelwal (2009) and Makled et al. (2019) have
reported extracellular enzyme production in
significant amounts because of presence of suitable
gut adherent enzyme producing microflora. The
specific enzyme activities were also found highest in
treatment D3 and lowest in control DC which may be
due to better dietary protein utilization or due to
colonization of probiotics bacteria and its exogenous
enzyme production. When probiotics supplementation
exceeds the optimum level, no further improvement in
growth performance and nutritive physiology of the
fish was observed, rather these parameters decreased.
This might be due to the fact that probiotics bacteria
incorporated in the feed might have competed
amongst themselves and their colonization was not
proper, resulting in the decline in exogenous/
extracellular enzyme production and thus low
digestibility, low growth and high feed conversion
ratio. These findings could be attributed to the specific
feature of probiotic bacterium which stimulate the
digestive system of host to increase the intestinal
enzymatic activities (Eslamloo et al., 2012; Bhatnagar
and Saluja, 2019) and inhibition of other harmful flora
along fish gut (Makled et al., 2019) thus resulting in
better growth performance of fish.
In aquaculture, water quality deteriorates mainly
205
Int. J. Aquat. Biol. (2020) 8(3): 194-208
due to accumulation of metabolic wastes such as
ammonia and orthophosphate excretion in the holding
water. Bacillus sp. reduces the quantity of ammonia
and nitrite in the water as it degrades the organic
matter and facilitates nutrients recycling (Skjermo and
Vadstein, 1999; Sanders et al., 2003). The findings of
Raparia and Bhatnagar (2016) and Bhatnagar and
Lamba (2017) showed that dietary supplementation of
B. coagulans and B. cereus, respectively, lowered the
excretion of total ammonia (N-NH4) and
orthophosphate (o-PO4), respectively. Similarly, in
the present study, B. coagulans supplementation at
3000 CFU g-1 improved the water quality parameters
and also reduced pathogenic bacteria load to
significant levels.
RBC and WBC increased in yellowtail infected
with N. kampachi (Ikeda et al., 1976). The result of
the present experiment also revealed an increase in
TLC and TEC counts in groups D2 and D3 compared
to the control (DC). This indicated the heightened
immune response in the fish fed on feed containing
B. coagulans, probably due to its immunostimulatory
effect. Similar findings were reported by
Bandyopadhyay et al. (2015), Makled et al. (2017) and
Bhatnagar and Dhillon (2019) where hematological
parameters i.e. TLC and TEC count showed
enhancement when fish were fed on probiotic
supplemented diet.
It has been shown that Bacillus strains
supplementation in diet could increase disease
resistance in fish through the stimulation of cellular
immune function, such as phagocyitc activity
(Merrifield et al., 2009). Phagocytosis is responsible
for early activation of the inflammatory response and
is mediated by phagocytic cells such as neutrophils,
monocytes and macrophages in fish (Kwak et al.,
2003). Significant increases of phagocytic activity
(PA) and phagocytic index (PI) was recorded in
E. coioides fed B. pumilus or B. clausii containing
diets for 60 days compared with those fed the control
diet (Sun et al., 2010). Sumathi et al. (2014) reported
that diets with B. megaterium and Pontibacter
inclusion induced highest phagocytic ratio and
phagocytic index in L. rohita. In present study also,
significant increase in PA and PI were found in
treatment D3 compared with those fed on control diet.
In line with our finding, Bandyopadhyay and Patra
(2004) found that isolated bacterium B. circulans PB7
could significantly improve the phagocytic ratio and
phagocytic index of C. catla (Ham.). Bhatnagar and
Dhillon (2019) in L. calbasu and Bhatnagar and Saluja
(2019) in C. catla have also reported high PI and PA
with high growth performance.
Zhou et al. (2010) confirmed the isolated probiotics
B. coagulans 16 from the gut of Oreochromis niloticus
enhances the immune and health status, thereby
improving growth performance which supports the
results of present studies for C. catla. However, they
used culture of probiotics as water additives where as
in present study probiotic bacterium was used as
dietary supplement.
In L. rohita fed with feed containing B. subtilis, the
survival rate after challenge with A. hydrophila was
significantly higher in the treatment group compared
to the control. Administration of yeast glucan
enhances the survival of carp infected with
A. hydrophila (Selvaraj et al., 2005). The per cent
mortality during challenge trial with A. hydrophila
was low in the groups fed with probiotic bacterial
strain B. coagulans @ 3000 CFU g-1. The survivorship
plot indicated that there is a significant difference
between the survivorship curves in each treatment;
similar plot has been reported by Bhatnagar and
Lamba (2017) and Bhatnagar and Dhillon (2019) in
their studies on C. mrigala and L. calbasu,
respectively. The high rates of establishment of
bacterium in the gastro-intestinal tract of fish treated
with B. coagulans have suppressed the A. hydrophila
infection, which ultimately resulted in the higher
survival in treatment D2 and D3 in present
investigation.
Acknowledgements
We are grateful to University Grants commission,
New Delhi, India for sanctioning support under
Special Assistance Programme at DRS-I.
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Bhatnagar and Raparia / Bacillus coagulans as probiotic bacterium and its dosage for Catla
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