Partha
Bandyopadhyay • Biplab Sarkar • Arabinda Mahanty • Raja M. Rathore • Bidhan Chandra Patra
Received: 29 December 2014 /
Revised: 23 April 2015 / Accepted: 20 May 2015 / Published online: 21 June 2015
The
National Academy of Sciences, India 2015
Abstract Bacillus sp. PP9, a
noble bacterial isolate of mrigal (Cirrhinus mrigala) gut was investigated for
its efficacy as a dietary probiotic against mrigal (C. mrigala) through a dose
dependent impact assessment. Mrigal fin-gerlings (avg.wt. 2.5 ± 0.20 g) were
fed with three dif-ferent doses (2 9 104,
2 9 105 and 2 9 106
CFU) of Bacillus sp. PP9 admixed with 100 g feed for a period of 60 days. It
was found that the feed with Bacillus concen-tration of 2 9 104
CFU exhibited significantly higher growth, maximum RNA DNA ratio, lower food
conversion ratio in comparison to other two feed types. Enhanced intestinal
protease and a-amylase activity followed by maximum hepatic glutamic
oxaloacetic transaminase and glutamate pyruvate transaminase levels were also
moni-tored in this specific feed type. Important hematological parameters like
hemoglobin percentage, total erythrocyte count, total leukocyte count,
corpuscular hemoglobin also indicated a healthy trend in this dose.
Furthermore, the
1
Aquaculture Research Unit,
Department of Zoology, Vidyasagar University, Midnapore 721102, West Bengal,
India
2
School of Biotechnology, KIIT
University, Bhubaneswar 751024, India
3 Nutrimar As,
7266 Kverva, Norway
4
Central Inland Fisheries
Research Institute, Barrackpore, Kolkata 700120, India
5
National Institute of abiotic
stress management, Baramati, Pune, Maharashtra, India
6
Present Address: Finray Biotech INC, Vedant Complex, NH NO. 8, Survey NO 552, Bapod, Near Hanumanji Temple, Waghodia Chowkdi, Barada – 390019. India.
same dietary treatment exhibited highest levels of total
serum protein, albumin globulin ratio and serum bacteri-cidal activity. The
growth, nutrition and immunological parameters showed a declining trend with
increase in the bacterial concentration as well as in the control group
(without probiotic). In summary, dietary supplement of 2 9 104/100
g appears as a potential probiotic dose of Bacillus sp. PP9 for growth,
nutritional efficacy as well as immunological modulation of C. mrigala and
denotes a recommended concentration for its future field applications.
Keywords
Bacillus sp PP 9 Feed formulations Cirrhinus mrigala Probiotics Growth
Introduction
In recent times, aquaculture appears as one of the fastest
growing animal food producing sector and is projected as efficient agricultural
subsidiary for its contribution in high revenue generation, rural livelihood
and employment cre-ation. But like other farming practices, aquaculture system
is also stressed with infections and diseases causing major obstacles to its
productivity and sustainability [1]. It is very
important to design quality health management systems in fish to improvise
disease resilience as well to recover from pathogenic infestation. In recent
years, applications of vaccine, antibiotics and probiotics are accepted
therapeutic tools to boost fish health. Among such methods, the pro-phylactic
use of probiotics has gained more attention [2]
due to its easy availability and handling, wide applicability as well as
ecological viability. One obvious reason for selecting probiotics is its unique
applicability at larval as well as early fry and fingerlings stages where
vaccines
cannot be delivered. At these stages, mortality is high due
to proliferation of opportunistic pathogens even at low initial infection
pressure [3]. Application of antibiotics has
been a matter of concern in contemporary times, due to emergence of resistant
varieties, environmental issues and export related regulations. In such cases,
alternative strategies like application of probiotics and other nutri-tional
interventions are very useful and being applied [4].
In last decade, an upsurge in
probiotics research has been observed and numerous trials have been conducted
with its supplements to improve health status and produc-tivity of farmed
animals. Appropriate probiotics applica-tions have shown an improvement in gut
microbial balance leading to enhanced food absorption [5]
and reduced pathogenic problems in gastrointestinal tract [6]. Though multiple probiotics species have been
screened and evalu-ated including Lactobacillus sp., Bacillus sp. and various
mixed cultures [4, 7–9] specially in economically impor-tant terrestrial
animals, yet reports on commercial appli-cation in aquaculture varieties are
less. Among them, Bacillus possesses important properties like adhesion,
immune-stimulation and bacteriocin secretion etc. as a potential probiotic
candidate [8]. However, endeavors to search for
new Bacillus strains with improved performance are still continued. Bacillus
sp. PP9 was isolated and screened from the gut of Cirrihinus mrigala and
thereafter, identified through molecular biology tools as a new pro-biotics
species [8]. The present study was aimed to
eval-uate the efficacy and performance of Bacillus sp.PP9 strain on growth,
nutrition and immunity of C. mrigala finger-lings, as a noble probiotic
candidate.
Material
and Mmethods
Fish
Sample Collection
Cirrihinus mrigala fingerlings were collected from Back-ery
Aqua Farm, a carp culture centre located near Midna-pore city, West Bengal
(22L250
N and 87L200
E), India. Fishes (Weight 2.5 ± 0.020 g; Length 7.1 ± 0.2 cm) were released
into continuous flow through aquaria (200 l capacity) and were acclimatized for
15 days in laboratory conditions. Before acclimatization, fish were dipped in
0.9 % potassium permanganate to dispel any external infections.
Preparation
of Experimental Feeds
Fish feeds were prepared using locally available
ingredi-ents including mustard oilcake (40 %), fish meal (40 %), rice polish
(13 %), tropica flour (5 %), vitamin and mineral mixture (1 %) and cod liver
oil (1 %). Feed were
formulated by ‘‘square-method’’ applying determined pro-tein
values of the ingredients. Dough was prepared with careful blending of
ingredients and the feeds were pelleted separately with ‘hand pelletizer’. The
pellets were dried in an oven at 38 LC to less than 10 % moisture and stored in
airtight containers at ambient temperature.
Preparation
of Experimental Diet
Probiotic bacterium Bacillus sp. PP 9, an isolate of C.
mrigala gut, was incubated in nutrient broth at 31 LC for
48
h. The bacterial cultures were
suspended in sterile saline water after washing with 1 % sodium chloride [4]. Experimental feed were prepared by absorbing
suspension of the mentioned probiotic strain. The suspended bacterial
cultures were sprayed over the earlier prepared feeds in the
concentration of 2 9 104
(M1), 2 9 105
(M2) and 2 9 106
(M3) Bacillus sp. CFUs per 100 g feed excluding the control
feed (MC). The feeds were kept at 4 LC inside vacuumed plastic containers and the
bacterial concentra-tion were monitored at a weekly interval.
Design
of Experimental Trial
Three aquaria (100 L capacity) were allotted for each
treatments as well as control group with 30 L water and constant aeration
facility. Fifteen fishes were kept in each aquarium. The physico-chemical
properties of water were monitored weekly as per recommended protocol of APHA
(2012) [10]. Fishes were fed two times a day
(morning and evening) at 4 % body weight and the leftover feeds were siphoned
out after 1 h of dispension into the aquarium. Fifty percent of the used water
was exchanged regularly with fresh water. Experiments were conducted at room
temperature for 60 days regime. The net weight was measured at every 15 days
and feed quantity was read-justed. Evaluation of the dietary performances were
con-ducted through estimation of nutritional indices like live weight gain
(LWG), average daily growth (ADG), feed conversion ratio (FCR), specific growth
rate (SGR) and protein efficiency ratio (PER) as per standard protocols [4]. At the end of experiment, three fishes from the
each treatment and control group were randomly selected and sacrificed with the
application of high dose anesthesia (MS222; Sigma Chemicals, India) and
different tissue parts (liver, intestine etc.) were collected for further
assessment.
Proximate
Analysis
Proximate analyses of feed ingredients and fecal matter were
performed according to the method of AOAC [11].
Crude protein and fat contents were estimated by micro-Kjeldahl and soxhlet
methods respectively. Crude fiber content of the
defated samples were determined following the protocol of
Patra et al. [12]. Ash content was determined
by incinerating the samples in muffle furnace (at 500 ± 50 LC for 10 h). For
fecal matter estimation, pooled fecal matter was collected into petri dishes
from aquarium bed by a pipette. The col-lected material was dried in oven at 55
LC and stored in air-tight containers until further analysis.
Biochemical
Analysis
After 60 days of trial period, DNA and RNA contents in the
liver were estimated following the method of Munro and Fleck [13]. The intestinal protease and a-amylase activities
were determined by the standard method prescribed by Bernfeld [14]. Briefly, the a- amylase activity was deter-mined
in triplicate by using 1 % soluble starch, as substrate, with
3,5-dinitrosalicyclic acid (DNS) at 550 nm. Specific a-amylase activity is
reported as U = lg of maltose min-1
- mg-1 of protein present in the
enzyme extract tested at 37 LC. Liver glutamic oxaloacetic transaminase (GOT)
and gluta-mate pyruvate transaminase (GPT) activities were deter-mined
following the method of Bergmeyer and Bernt [15].
Hematological
Assays
At the end of 60 days trial, blood samples were collected
through cardiac puncture before sacrificing the fishes for other assays and collected
into heparinised vials. Hema-tological parameters were estimated according to
the method of Decie and Lewis [16]. The total
serum protein (TSP) and albumin contents were estimated following the method of
Lavanya et al. [17]. The globulin content was
calculated as the difference between the total proteins and the albumins.
Immunological
Assays
At the end of feeding trials, fishes from the experimental
aquaria of each group were bled to collect serum samples and analyzed for
agglutination titre using microtitre plates [18].
Collected sera were stored at -0.20 LC until further analysis. Serum
bactericidal activity was estimated by the prescribed method of Kajita et al. [19].
Above all, major water quality
parameters were main-tained in ambient range during the entire experimental
period (Temperature-28 ± 3.25 LC; pH-7.48 ± 0.21; Total alkalinity (ppm)-140.25
± 5.6; Dissolved Oxygen (ppm) -4.6 ± 0.42; Total ammonia (ppm) -0.13 ± 0.02.
Statistical
Analysis
Descriptive statistics was employed to analyze the
experi-mental data by using the SPSS 17. For evaluating the
significant differences (p \ 0.05) in the treatments of
dietary performances, nutritional indices, enzymatic activities, RNA DNA ratio
and immunological parameters, Duncan multiple Range Test (DMRT) were performed.
Results
and Discussion
In aquaculture, probiotics have captured special attention
for its efficacious function for supplementary synthesis of digestive enzymes [20], and improvement in growth per-formance of the
farmed species [21, 22].
Bacillus have been widely used as putative probiotics for its beneficial
nutritional impact, antagonism against microbial pathogens and easy
supplementation [23, 24],
as reported for larval development of Gilthead sea bream (Sparus aurata, L.) by
Arıg et al. [25]. The purpose of current
experiment was to evaluate the efficacy of Bacillus sp. PP9 on growth,
nutrition and immunological status in C. mrigala at an effective dose.
Proximate
Analysis of Feed and Fecal Matter
The proximate composition of prepared feed, its ingredi-ents
and concentration of the probiotics are presented in Fig. 1. All the prepared feed were isocaloric and
equivalent in terms of P/E ratio and nitrogen free extract (NFE) content. As
shown in Fig. 1a, the proximate composition of
ingredients applied in preparing experimental feeds were quite different. The
crude protein percentage of rice polish, mustard oil cake, and fish meal was
13.10, 39.20 and 49.16, whereas the crude lipid percentage was 5.22, 11.15, and
6.58 respectively. The percentage of average crude protein on dry matter basis
was 36.90 whereas the crude lipid percentage was around 9.82 (Fig. 1b). Bacillus concentra-tion of three different
probiotic feed formulations are pre-sented in Fig. 1c.
Proximate analysis of the fecal
matter of different treatments and control group showed significant difference
in the protein, fat and ash content (Fig. 2).
Fecal matter proximate analysis revealed significantly (p B 0.05) higher
nitrogen excretion in fish fed with MC (13.15 ± 0.040 %) and least in fish fed
with M1 (12.41 ± 0.050 %). The crude lipid content varied between 3.64 ± 0.011
% (M1) and 3.98 ± 0.012 % (MC).
Fish
Growth and Nutrition Analysis
In relation to all the feeding regimes, growth of C. mrigala
was observed highest (p B 0.05; DMRT) in the M1 group (5.68 ± 0.018 g), while
lowest (5.37 ± 0.022 g) in MC. Growth in terms of weight gain percent was
significantly higher (127.20 ± 0.532 %) in M1 fed fish and least in MC
(114.80 ± 0.460 %) (Table 1).
In addition, highest SGR, PER and lowest FCR were observed in M1 fed C.
mrigala, whereas reverse trends were observed in MC fed fishes. M2 and M3 fed
groups showed an intermediary trend in per-formance between M1 and MC in a dose
dependant manner where M2 performed better than M3 (Table 1).
In the present study, higher
growth was noted in C. mrigala fed with M1 as evaluated by weight gain
per-centage (WGP), SGR, when compared with other probiotic treatments as well
as control. This result focuses on important inference that the probiotic
concentration of
2
9 104
per 100 gm feed, might be supportive for optimum dietary utilization due to a
positive correlation between the WGP and protease activity. Similar findings
were reported earlier by using particular concentration of Bacillus sp. S11
Table 1 Initial and
final body weight, live weight gain, weight gain percentage, FCR, SGR and PER
of Cirrhinus mrigala after 60 days trial period (values with the same letter
are not significantly different among treatments at 0.05 level)
Feeds
|
Fish weight (g)
|
Live wt. gain (g)
|
Weight gain (%)
|
FCR
|
SGR
|
PER
|
|
Initial
|
Final
|
||||||
MC
|
2.5 ± 0.020
|
5.37 ± 0.022
|
2.87 ± 0.009
|
114.80 ± 0.460a
|
1.11 ± 0.005a
|
1.27 ± 0.006a
|
1.93 ± 0.008a
|
M1
|
2.5 ± 0.015
|
5.68 ± 0.018
|
3.18 ± 0.012
|
127.20 ± 0.532b
|
1.02 ± 0.004b
|
2.06 ± 0.010b
|
2.13 ± 0.008b
|
M2
|
2.5 ± 0.012
|
5.60 ± 0.014
|
3.10 ± 0.007
|
124.00 ± 0.520c
|
1.02 ± 0.005b
|
1.34 ± 0.006c
|
1.82 ± 0.007c
|
M3
|
2.5 ± 0.018
|
5.54 ± 0.015
|
3.04 ± 0.010
|
121.60 ± 0.348d
|
1.03 ± 0.004d
|
1.32 ± 0.006c
|
1.33 ± 0.006d
|
in Penaeus monodon [26]. FCR
and PER are reported to be decreased with increasing protein contents [27] and the effects may differ with the species. In
the current study, although all the feeds were isonitrogenous but the
con-centration of probiotics in M1 feed might have contributed for maximum
nutrient utilization. Lesser nitrogenous con-tent in egestion were observed in
M1 fed groups which can be attributed to suitable probiotic concentration. This
is also obvious from the results that greater nitrogenous egestion was recorded
in MC fed fishes that exhibited a comparatively low feed utilization.
Biochemical
Analysis
As represented in Fig. 3,
maximum RNA DNA ratio (1.38 ± 0.005) was registered in the fish fed with M1
followed by M2, M3 and the lowest (1.16 ± 0.006) in the MC group. RNA DNA ratio
is an important indicator of growth [27]. The
ratio was highest in the M1 with higher dietary utilization and growth
performances. Zehra and Khan [28] reported an
enhanced RNA DNA ratio through dietary lysine enrichment in catla (Catla catla)
fingerlings. The results of present study are highly corroborative with such
findings where supplement of 2 9 104
Bacillus sp.
PP
9 cells 100 g-1
of feed (M1) projected better RNA DNA ratio and growth.
Intestinal protease and a
amylase activities were sig-nificantly highest (p B 0.05) in M1 fed group,
whereas lowest in MC fed fishes (Fig. 4a, b).
Hepatic GPT and GOT levels were observed significantly higher in M1 fed fishes
as compared to the group fed with MC (Fig. 4c).
Earlier reports revealed the fact that the activity of protease enhances with
high dietary protein [29], but in this
experiment all feeds were iso-nitrogenous and M1 showed greater protease
activity that might be related to greater dietary protein utilization.
Intestinal mucosa is one of the origin of carbohydrate digestive enzymes in
fish and the amylase activity is correlated with the carbohydrate content
Fig. 3 RNA DNA ratio in fishes fed with different feeds.
Different letters (a, b, c, d) above the bars corresponds to statistical
difference (p \ 0.05)
of the diet [30]. Hence, M1
fed fishes exhibited greater a amylase activity which indicates greater carbohydrate
uti-lization. The present study also showed greater a-amylase activity in all
test feeds as compared to initial activity. These results indicate the efficacy
of other probiotic con-centrations to utilize the carbohydrate content of the
diet. Hepatic GOT and GPT activity in fish also increased in probiotics
supplemented diet over the initial level. The highest level of GOT and GPT in
M1 group signifies better utilization of dietary protein. Within the protein,
most of the amino acids undertake transamination reactions and transaminases
are primarily located in both mitochondria and cytosol of a cell are induced by
high protein diet [31]. Thus a positive
correlation between hepatic GOT, GPT level and dietary protein could be
extrapolated. In other way, M2 and M3 fed groups showed a dose dependant
declination in DNA:RNA, intestinal protease and a amy-lase activity and hepatic
GOT and GPT levels but showed better activity than in control.
Hematological
and Immunological Assays
Hematological parameters such as total leucocyte count
(TLC), total erythrocyte count (TEC), haemoblobin (Hb), hemotcrit (Hct), mean
corpuscular hemoglobin (MCV), mean corpuscular volume (MCH), and mean
corpuscular volume hemoglobin (MCHC) of C. mrigala were signifi-cantly high (p
B 0.05) in M1 fed fishes in comparison with other treatments (Table 2). Impacts of different feeding trials on albumin
globulin ratio was observed maximum at MC fed fishes (1.890 ± 0.008) and minium
in M1 (1.545 ± 0.006) (Fig. 5). The impacts of
different probi-otics on non-specific and specific immunity were recorded
significantly high (p B 0.05; DMRT) for NBT positive values (63.33 ± 0.326),
antibody titre (125.76 ± 5.490) and lowest bactericidal activity (7.23 ± 0.010
9 103 cells ml-1)
in M1 treated C. mrigala (Fig. 6).
As a liquid connective tissue,
blood functions as a patho-physiological indicator and measuring blood
parameters are important for diagnosing fish health status. In the current
evaluation, blood parameters retained the optimum range in all the doses [32] and M1 fed fishes indicated the best positive
impact. Statistically significant increase was recorded in all the parameters
of non-specific and specific immunity. Highest NBT positive cells, anti-body
titre (p B 0.05; DMRT) and lowest bactericidal activity, albumin globulin ratio
was obtained in M1 fed fishes and these phenomenon could have been undertaken
due to better lymphocyte proliferation and consequent immunoglobulin synthesis
in fish. These results are cor-roborated with the report given by Iwashita et
al. [33] in which three candidate probiotic
species used were Bacillus subtilis, Saccharomyces cerevisiae and Aspergillus
oryzae.
Table 2 Comparative
hematological parameters of Cirrhinus mrigala after 60 days of feeding trial
(Figures having different letter (super-scripted) in the same row are
significantly different at 0.05 level)
Parameters
|
Treatments
|
|||||||
MC
|
M1
|
M2
|
M3
|
|||||
TEC (9 106 mm3)
|
1.94
|
± 0.004a
|
2.43
|
± 0.002b
|
2.11
|
± 0.009c
|
2.15
|
± 0.010d
|
TLC (9 103 mm3)
|
19.8
|
± 0.011a
|
28.8
|
± 0.001b
|
25.2
|
± 0.006c
|
23.9
|
± 0.007d
|
Hb (g %)
|
9.7
|
± 0.004a
|
12.4
|
± 0.006b
|
10.9
|
± 0.002c
|
11.2
|
± 0.002c
|
Hct (%)
|
29.8
|
± 0.023a
|
33.4
|
± 0.016b
|
30.8
|
± 0.011c
|
30.9
|
± 0.009c
|
MCV (lm3 cell-1)
|
153.608
|
± 0.163a
|
137.448
|
± 0.094b
|
145.971
|
± 0.306c
|
123.107
|
± 0.247d
|
MCH (pg cell-1)
|
50.000
|
± 0.023a
|
51.028
|
± 0.056b
|
48.047
|
± 0.073c
|
44.621
|
± 0.062d
|
MCHC (g 100 ml Hct-1)
|
32.550
|
± 0.068a
|
37.125
|
± 0.022b
|
35.389
|
± 0.049c
|
36.254
|
± 0.086d
|
NBT positive cells (%)
|
46.33
|
± 0.381a
|
63.33
|
± 0.326b
|
61.66
|
± 0.201c
|
48.66
|
± 0.406d
|
From multiple correlations, it was also noticed that RNA DNA
ratio was negatively correlated (p B 0.01) with albumin globulin ratio and
positively correlated (p B 0.05; DMRT) with antibody titre. Results of current
investigation suggests that the concentration of probiotic Bacillus sp. PP9 in
feed M1 was highly efficacious on improving the overall physiological and
immunological status of C. mri-gala fingerlings.
In the current experiment,
other two Bacillus concen-trations (M2) and (M3) also exhibited higher trends
in growth, nutrition and immunological parameters in respect to control, but
their performances were not the best. The dose dependent data obtained from the
experiments proved the accuracy of selecting M1 as best probiotic candidate.
Similar dose dependant efficacy study were also conducted by Bandyopadhay et
al. [34] in applying S. cerevisiae as a feed
supplemented probiotics in rohu (Labeo rohita).
Conclusion
The present investigations demonstrated the role of noble
bacterium, Bacillus sp. PP9 to promote enhanced growth and immunity in Indian
major carp, C. mrigala as a dietary probiotics. Inclusion of Bacillus sp.PP9 at
a concentration of (2 9 104/100
g feed) showed highest efficacy. 2 9 104/100
g of Bacillus dose also revealed the critical point of best performance and
surpassing this point would initiate inhibition of above mentioned activities
in C. mrigala. But further trials should be conducted in pond conditions before
its commercialization.
Acknowledgments Authors would like to express their thanks
to Indian Council of Agricultural Research (ICAR), New Delhi, India for
financial assistance. The authors are also thankful to Head of the Department,
Department of Zoology and Vice-Chancellor, Vidyasa-gar University for providing
necessary facilities.
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