Control of bacterial pathogens in fish farms has been routinely achieved by
administration of antibiotics and chemotherapeutants. However, these were less
successful due to the emergence of drug resistant strains (Thyssen
and Ollevier, 2001). As a result, the modern aquaculture industry demands
alternative prophylactics that may help to keep a healthy environment resulting
in better production and higher profits. Within this context immunostimulants
or probiotics seem to be very promising alternative (Balcazar
et al., 2007). The research on probiotics for aquatic animals is
increasing with demand for an eco friendly aquaculture (Verschuere
et al., 2000).
Most of these studies have been related to challenge trials and suppression
of pathogen by probiotics (Vaazquez et al., 2005)
while another promising aspect is the use of probiotics in fish diets for potential
improvement in feed efficiency.
Buts et al. (1999) have reported that in terrestrial
animals the probiotics influence digestive processes by increasing the beneficial
microbes thereby increasing microbial enzymatic activities and digestive enzyme
activities. This results in higher digestibility of food and improved feed utilization
(Bomba et al., 2002) and similar effect has been
reported in some aquatic animals such as turbot larvae (Gatesoupe,
1991) and shrimps (Uma et al., 1999). Aquaculture
diets are conventionally based on expensive feed stuffs such as fish and fish
meal. Development of aquaculture will be greatly enhanced by finding alternative
and less expensive ingredients. The culture of murrel Channa striatus
is a very promising industry in Asian countries like India but the most serious
constraints are non availability of seeds and lack of knowledge of feeding techniques.
Murrels are carnivorous, piscivorous and cannibalistic in nature. Feeding is
a herculean task and hence fish farmers are facing problems due to non-availability
of readymade feed. At this juncture the nutritional physiology of murrels is
very much important to promote murrel culture among farms (Ghosh
et al., 2002).
Efinol® FG, a commercial probiotic is a combination of highly
concentrated thermostable beneficial microbes (Bacillus subtilis, B.
coagulans and Saccharomyces cerevisiae along with selected nutrients,
free flow and anti caking) that can withstand and high temperatures and function
as growth promoters and immunomodulators. Efinol® FG is non-pathogenic,
non-toxic and can survive in gut and remain stable and viable for long periods
under storage and field conditions. Semi moist feeds are recently used in grow
out culture of C. striatus because of their high feed conversion efficiency,
easy preparation, digestion and better conversion ratio (Haniffa
et al., 2002).
This study was intended to determine the effect of Efinol® FG on growth, feed efficiency, microbial community and enzymatic activities of the murrel C. striatus.
MATERIALS AND METHODS
C. striatus fingerlings (2.7±0.03 g) were collected from Centre for Aquaculture Research and Extension (CARE), Aquafarm. The fingerlings were randomly selected and distributed in cement tanks (1000) filled with water at a rate of 45 fingerlings per tank. Total 3 Replicates were maintained for each of the 5 treatments.
About 5 diets were prepared an unenriched control diet and four diets containing
Efinol® FG. Efinol® FG was obtained from Bentoli
AgriNutrition (USA). Lyophilized cells [1011 Colony Forming Units
(CFU) g-1 of cells wet weight] were maintained at -20°C prior
to use at a concentration of 103 CFU g-1 (T1), 104
CFU g-1 (T2), 105 CFU g-1 (T3) and 106
CFU g-1 (T4) (Table 1). The ingredients were mixed
and the probiotics Efinol® FG was added to the autoclaved semi
moist feed and kept at -20°C. The biochemical analysis of the feeds was
analyzed by standard methods (AOAC, 1995). The fingerlings
were fed 4% of their body weight thrice a day for 45 days. About 13rd of the
water was changed daily. The temperature 26-28°C, salinity 27-28%, Total
ammonia 0.02 mg L-1 and pH 7-7.3 were recorded. Fingerlings were
weighed at 15 day intervals to determine weight gain, Specific Growth Rate (SGR),
Food Conversion Ratio (FCR) and survival rate:
Gut samples were well homogenized and serial dilution was performed under aseptic
conditions, Total heterotrophic bacterial counts were recorded using Tryptic
soy Agar plates. Selective media such as Sporulating agar (B. subtilis),
Alkaline Bacillus media (B. coagulans) and Potato Dextrose Agar (yeast)
were used and the observations were recorded as colony forming unit (CFU mL-1).
|| Proximate composition of selected ingredients
|aMineral premix to supply the following elements
(mg kg-1 diet): zinc (as sulphate) 72, iron (as sulphate), 36,
manganese (as sulphate) 12, copper (as sulphate) 24, cobalt (as chloride)
0.6, iodine (as iodate) 1.2, chromum (trivalent as chloride) 0.8, selenium
(as selenate) 0.2 and molybdenum (as molybdate) 0.2; bVitamin
premix: (mg kg-1) vitamin B12, 0.1; nicotinic acid, 80.0: riboflavin,
50; pantothenic acid, 180; menadione, 40 folic acid, 6.0; biotin, 0.6; thiamin
hydrochloride, 15; pyridoxine, 60; thiamin, 40; inositol, 400; astaxanthin,
60; choline chloride, 20.0; vitamin C, 250 and (IU) vitamin A, 6000; vitamin
D3, 2000; vitamin E, 6000 IU
Protease activity was evaluated using Lowry et al.
(1951) amylase activity following Jiang (1982) and
Worthington and WBC (1993) and expressed as specific activity
(U mg-1 protein)and lipase activity based on methods of Borlongan
(1990) and Jin (1995) and expressed as U g-1.
Means and standard deviations were compared by one-way ANOVA. Duncans
multiple range test using SPSS (version 7) software was performed to find significant
(p<0.05) differences in growth parameters.
RESULTS AND DISCUSSION
The total heterotrophic count in the initial fish was 5.4x104 CFU g-1 and increased to 7.7x108 CFU g-1 in the T4 fish with 106 CFU g-1. B. subtilis, B. coagulans and S. cerevisiae count was determined using their respective selective media. The total B. subtilis in Sporulating agar was 9.3x105 CFU g-1. B. coagulans in alkaline bacillus medium was 9.5x105 CFU g-1 and S. cerevisiae in potato dextrose agar medium was 9.7x104 CFU g-1 (Table 2).
The total heterotrophic bacterial count differed significantly among Efinol®
FG diets. When compared to control C. striatus fed diet with Efinol®
FG in different concentrations showed better survival in Table
3. The fish readily accepted all 5 diets. The control fish had statistically
lower growth and survival than fish fed with probiotics (Efinol®
FG) enriched diets. No mortality was recorded in T4 group whereas in control
C. striatus 12% mortality was shown in Table 3. After
45 days, there was a significant difference between the mean weights of treated
C. striatus (T1-T4). The highest weight gain of 16.37±0.08 g was
noticed in T4 (106 CFU g-1). The weight gain of test fish
of all the treatments was significantly higher than that of the control (11.59±0.17
||Growth of total heterotrophic count and B. subtilis,
B. coagulans, S. cerevisiae in selective media. All values are reported
as CFU g-1 and ±indicates the standard deviation
|| Growth performance of C. striatus fed with experimental
|The mean values having different superscripts in the same
row are significantly different at p<0.05%, level and ±indicates
the standard deviation
Values obtained for weight gain (12.4-16.4 g) and specific growth rate (3.8-4.2)
of all probiotic treated groups were also significantly higher (p<0.05) than
those of the control (11.59 and 3.66 g). Mean values of Weight Gain (WG) and
SGR were significantly different (p<0.05) among the different treatment groups.
The highest SGR was observed in T4 (4.22±0.09% day-1) where
the FCR was also the least (1.28±0.09%).
After 45 days of the experimental tenure the digestive enzyme activity of all trial groups showed significant difference when compared to that of the control. But there was no significant difference between T1 and T2 trials. The Protease activity was significantly higher (135.26±14.15 U mg-1) in T4 followed by T3 (125.80±6.16 U mg-1), T2 (119.35±6.13 U mg-1) and T1 (109.44±5.23 U mg-1). In the control the same was measured as 93.75±4.16 U mg-1.
The average value of Amylase activity was significantly higher (68±5.23 U mg-1) in T4 when compared to T1 (49.97±3.98 U mg-1) and the control (47.83±3.75 U mg-1). Similarly, the average value of lipase activity in the intestine of T4 C. Striatus was also higher (95.78±7.23 Ug-1) (Fig. 1).
The probiotics (Efinol® FG) feed fed fishes exhibited superior
growth performance in comparison to the control feed fed fishes. Similar, results
have been reported in Seabass (Dicentratrchus labrax) by Carnevali
et al. (2006) and in Indian major carps by Ghosh
et al. (2003) and Swain et al. (1996)
and in live bearing ornamental has fish (Ghosh et al.,
2007). Among the different probiotics diets T4 diet showed comparatively
better growth performance than others. This finding corresponds well with earlier
studies by El-Haroun et al. (2006) who found
that commercial probiotics Biogen® in higher concentration resulted
in better growth performance in Oreochromis niloticus. They observed
higher SGR and optimum FCR in Biogen® incorporated feed. Ahilan
et al. (2004) has also concluded that the application of probiotics
showed higher SGR and survival in gold fish (Carassius auratus).
The composition of intestinal microbiota is highly variable depending on the
developmental stage and the environmental conditions (Ringo
and Birkbeck, 1999; Huber et al., 2004).
In the present study the final microbial count was highest in probiotics fed
fishes in particular T4 diet fed fishes.
The results were supported by Ramakrishnan et al.
(2008) and Wache et al. (2006) who report
increased bacterial count in common carp (Cyprinus carpio) and rainbow
trout fed with probiotics diets. Similarly, probiotics promoted colonization
of bacteria in the fish gut for a prolonged period and had capacity to adhere
and grow well in vitro in the intestinal mucus from turbot (Makiridis
et al., 2000). The probiont (B. subtilis, B. coagulans
and S. cerevisiae) load was found higher in T4 diet. Similarly, Robertson
et al. (2000) observed a constant increase in Probiont (Carnobacterium
sp.) population in the gut of rainbow trout and Atlantic salmon fingerlings
fed with probiotic diet.
||Specific activity of protease, amylase and lipase in intestine
content of C. striatus fed with control and four experimental diets
containing probiotic Efinol®FG. Control, T1 Efinol®
FG 103, T2 Efinol® FG 104, T3 Efinol® FG 105,
T4 Efinol® FG 106 at the end of 45th day. Means with different
superscripts were significantly different (p<0.05)
As supplementary components in aquaculture feeds, probiotics have strong adhesive
and growth abilities (Mukhopadhyay and Paul, 1996).
It can therefore, be inferred that Efinol® FG is effective in
viably colonizing and proliferating in the digestive tract of host.
The effect of probiotics supplementation at intestine level was evaluated by
the activity of enzymes viz, protease, amylase and lipase. In the present study,
the enhanced nutrient and enzyme levels in by probiont addition led to increased
food digestion and absorption which in turn led to better growth of the fishes
ingesting the probiotics cells. A possible explanation proposed by procedure
by several researchers is that probiotics actively procedure a range of relevant
enzymes such as amylase, protease and lipase (Fuller, 1989;
De Schrijver and Ollevier, 2000; El-Haroun
et al., 2006) and stimulates the specific and total activities was
found to be higher in T4 diet. Similarly, the increase in digestibility by enzyme
activities through the use of probiotics be demonstrated in white Shrimp (Litopenaeus
vannamei) by Lin et al. (2004), Rodriganez
et al. (2009) in juvenile Senegalese sole (Solea senegalensis)
and Ghosh et al. (2002) in rohu (Labeo rohita).
In the study, the findings showed that the commercial probiotics Efinol® FG (106 CFU mL-1), when compounded with feed improved the growth performance and survival of murrel C. striatus. The increase in specific activities of enzymes coupled with the substitution of pathogenic microbes by beneficial probiont population in the intestine of probiotics feed fed fishes led to enhance of food which in turn contributed to the improved survival and growth of C. striatus.
Researchers would like to thank the Principal Rev. Dr. Alphonse Manickam S.J for providing necessary facilities for this research in St.Xaviers College, Palayamkottai, Tamilnadu and Dr. Victor Suresh for providing a Jovika Media Private Ltd., project.