Efficacy of Probiotics on Survival , Growth , Biochemical Changes and Energy Utilization Performance of Macrobrachium rosenbergii ( De Man 1879 ) Post-larvae

The efficacy of combined probiotics, Lactobacillus sporogenes and Saccharomyces cerevisiae (LS+SC) on survival, growth, biochemical changes and energy utilization of Macrobrachium rosenbergii post larvae (PL) was examined. Each probiotic organism was individually tested at four different concentrations (1-4%) separately. The best concentration in each probiotic species was combined and tested for its suitability in aquaculture usage. The basal diet was incorporated with probiotics, LS+SC (4:4) at five different concentrations 0% (control), 1%, 2%, 3% and 4%. These diets were fed to M. rosenbergii PL for a period of 90 days. After the feeding trail, 2% LS+SC incorporated diet had significantly (P<0.05) higher survival, WG, SGR, FCE and PER compared with other experimental groups than the control. Whereas, the FCR was significantly (P<0.05) lower in 2% LS+SC incorporated diet fed PL. Similarly the proximate composition of the protein, amino acid, carbohydrate, lipid and ash content were significantly (P<0.05) higher in 2% LS+SC incorporated diet fed PL than the control. The energy utilization parameters were significantly (P<0.05) higher in 2% LS+SC incorporated diet fed PL than the control. This study indicated that combined probiotics, LS+SC incorporated diets were beneficial for M. rosenbergii in terms of increasing growth and enhancing energy utilization performances.


Introduction
The giant river prawn, Macrobrachium rosenbergii is one among crustaceans, native to Southeast Asia, South Pacific countries, northern Oceania, and Western Pacific islands.M. rosenbergii has become the main freshwater prawn species for small-scale and largescale farming because of its fast growth, large size, better meat quality, omnivorous feeding habit and established domestic and export markets in Asia [1].A probiotic is generally defined as a live microbial food supplement, which improves the balance of the host animals' intestinal flora [2].The use of probiotics in the aquatic organisms is increasing with the demand for more environment-friendly aquaculture practices [3].A number of studies have shown that probiotics can improve the survival, growth and biochemical changes of the freshwater prawn M. rosenbergii [4][5][6][7][8][9][10][11][12][13].Therefore, the present study was undertaken to determine the effect of feeding combined probiotics, Lactobacillus sporogenes and yeast Saccharomyces cerevisiae on the survival, growth, biochemical changes and energy utilization performance of the freshwater prawn M. rosenbergii PL.

Diet preparation
The composition of the experimental diets is given in Table 1.The probiotics, L. sporogenes (Uni-Sankyo Ltd, Maharashtra, India) and yeast S. cerevisiae (Intercare Ltd, Gujarat, India) one gram of lyophilized powders contains 15x10 7 and 10x10 7 cfu cells respectively.The probiotics, LS+SC (4:4) were incorporated in to the test diets at five different concentrations individually 0% (control), 1%, 2%, 3% and 4% respectively.Diet formulation was done basically by ''Pearson's square-method'' using determined values of 40% protein content (Table 1).The proportion of each ingredient required was calculated precisely providing allowance for the premix.The dough was steam cooked and cooled to room temperature.After that different concentration of LS+SC (4:4) was mixed with the dough and the diets were pelletized separately with a locally made (Kolkata, India) hand pelletizer.The pellets were dried in a thermostatic oven (M/s Modern Industrial, Mumbai, India) at 40 0 C until it reached constant weight and stored in airtight jars at room temperature.

Feeding experiment
M. rosenbergii (PL-30) with the length and weight range of 1.54±0.02cmand 0.22±0.03grespectively were used for feeding experiment.40 PL for each diet in triplicate were maintained in plastic tanks with 20 L water.The PL was maintained at the stocking density of 2/l.One group served as control, which devoid of probiotics (0%).The experimental groups were fed with the respective concentration of LS+SC (4:4) incorporated diets.The feeding was adjusted to two times a day (6:00 am and 6:00 pm).The daily ration was given at the rate of 10% of the body weight of PL with two equal half throughout the experimental period.The unfed feed, faeces and moult if any, were collected after the respective hours of feeding.The feeding experiment was prolonged for 90 days; mild aeration was given continuously in order to maintain the optimal oxygen level.

Growth study
After the feeding trial, the growth parameters such as survival (S), weight gain (WG), specific growth rate (SGR), feed conversion rate (FCR), feed conversion efficiency (FCE) and protein efficiency rate (PER) were individually determined by following equations [6].Survival (%) = Total no. of live animals/Total no. of initial animals × 100 Weight gain (g) = Final weight (g) -Initial weight (g) SGR (%) = log w 2 -log w 1 /t ×100 (where, w 1 and w 2 = Initial and final weight, respectively (g), and t = Total number of experimental days) Feed conversion rate (g) = Total feed intake (g)/ Total weight gain of the prawn (g) Feed conversion efficiency (%) = Biomass (g)/ Total Feed intake (g) × 100 Protein efficiency rate (g) = Total weight gain of PL (g)/Total protein consumed (g)

Energy utilization study
The energy content of whole prawns, feeds, moult and faeces was measured using Parr 1281 Oxygen Bomb Calorimeter.The energy budget was calculated using the equation (C = (P g +E) + R + F +U) derived by Petrusewicz and Macfadyen [14]; where, C is the energy consumed in food; P g is the growth; R is the material lost as heat due to metabolism; F is the energy lost in faeces; U is the energy lost in excretion and; E is the energy lost in exuvia.

Biochemical constituents of the experimental animals
The initial and final day of the experiment, the biochemical constituents of the experimental animals were determined.The biochemical constituents, such as total protein [15], amino acid [16], lipid [17], carbohydrate [18], ash and moisture contents [19] of individual diet fed prawns were measured.

Microbial study
Microbial analyses [19], and yeast isolation [20] were performed in the rearing (control) water, control PL gut and experimental PL gut.

Statistical analyses
The data obtained in the present study were subjected to different statistical interpretations.One way analysis of variance (ANOVA; SPSS, 13.0) was used to determine whether significant variation between the treatments existed.Differences between means were determined and compared by post hoc multiple comparison test (DMRT).All the tests used a significance level of P<0.05.Data are reported as mean± standard deviations.

Morphometric data
Table 2 represents the morphometric data of LS+SC (4:4) supplemented diet fed PL group.The initial average body length and weight of PL was 1.54±0.02cmand 0.22±0.03g,respectively.After 90 days of feeding experiment, the final length and weight were found to be higher in PL fed with 2% LS+SC incorporated diet followed by the other experimental groups such as 3% LS+SC, 4% LS+SC and 1% LS+SC diets than the control.These differences were found to be statistically significant (P < 0.05).Similar improved morphometric data (length and weight) has been reported in M. rosenbergii PL fed with L. sporogenes bioencapsulated diets [5], Bacillus spp KKU02 and Bacillus spp KKU03 supplemented diets [11] and in shrimp, Penaus indicus after feeding with L. acidophilus, S. cremoris, L. bulgaricus-56 and L. bulgaricus-57 incorporated diets [21], Bacillus sp incorporated diets [22], Bacillus spp incorporated diets [23], B. subtilis UTM 126 incorporated diets [24].

Survival performance
Table 2 also depicted the survival performance of LS+SC (4:4) incorporated diet fed PL group.After 90 days of feeding trail, the survival performance of M. rosenbergii PL was found to be higher in 2% LS+SC incorporated diet.But it was only 80.00% in control diet fed prawn PL.On the other hand, the survival percentage of other experimental diets fed prawns was in the order of 1% LS+SC, 3% LS+SC, 4% LS+SC.These differences were found to be statistically significant (P<0.05).Similar improved survival has been reported in M. rosenbergii PL fed with L. sporogenes bioencapsulated diets [5], Binifit TM supplemented diets [6], Biogen® supplemented diets [8] and different probiotics supplemented diets [9], and in shrimps, fed with L. acidophilus, S. cremoris, L. bulgaricus-56 and L. bulgaricus-57 incorporated diets [21], B. subtilis incorporated diets [25] and Bacillus sp incorporated diets [26].

Growth performance
The results on growth performance of LS+SC (4:4) incorporated diet fed PL group are also displayed in Table 2.At the end of the feeding trail, the weight gain, specific growth rate, feed conversion efficiency and protein efficiency rate were significantly (P<0.05)higher in probiotics LS+SC (4:4) incorporated diet fed groups than the control.The result on weight gain, specific growth rate, feed conversion efficiency and protein efficiency rate were found to be maximum in 2% LS+SC incorporated diet fed prawn PL, followed by 3%, 4% and 1% when compared with control.The differences between control and experimental diets fed prawns were statistically significant (P<0.05).The feed conversion ratio was found to be higher in PL fed with control diet, followed by the PL fed with 1%, 4%, 3% and 2% diets.These differences were found to be statistically significant (P<0.05).Similar results have been reported in M. rosenbergii fed with L. sporogenes supplemented diets [4], bio-encapsulated L. sporogenes [5], Binifit TM incorporated diets [6], Bacillus spp supplemented diets [7], Biogen® incorporated diets [8], different probiotics supplemented diets [9], Bacillus spp KKU02 and Bacillus spp KKU03 incorporated diets [11], bio-encapsulated B. subtilis [10], Saccharomyces cerevisiae and yeast derivatives supplemented diets [27], bio-encapsulated L. acidophilus and L. sporogenes [12], bio-encapsulated L. ceremoris [13] and in fishes, L. plantarum and B. megaterium supplemented diets [28] Lactobacillus acidophilus and yeast Saccharomyces cervisiae incorporated diets [29], B. subtilis, B. licheniformis and Enterococcus faecium incorporated diets [30], bio-encapsulated L. casei [31], Bacillus spp supplemented diets [32], B. subtilis supplemented diets [33] and B. toyoi and B. cereus incorporated diets [34].

Biochemical constituents of experimental animals
The results on biochemical constituents, such as protein, amino acid, carbohydrate, lipid, ash and moisture content of LS+SC (4:4) incorporated diet fed PL group are also showed in Table 2.After the feeding trail experiment of 90 days, the protein, amino acid, carbohydrate, lipid and ash contents were found to be maximum in PL fed with 2% LS+SC diet, followed by the PL fed with 3%, 4% and 1% diets when compared with control.These differences were found to be statistically significant (P<0.05).In the case of moisture content just the reverse was recorded.A similar result in biochemical composition was previously observed in M. rosenbergii PL fed with L. sporogenes supplemented diet [4], bio-encapsulated L. sporogenes [5], Binifit TM supplemented diets [6], Biogen® incorporated diets [8] and bio-encapsulated L. sporogenes and L. acidophilus [12] and in fishes Lactobacillus acidophilus, and the yeast Saccharomyces cerevisiae supplemented diets [35], B. subtilis NIOFSD017 and L. plantarum NIOFSD018, and yeast S. cerevisiae NIOFSD019 incorporated diets [36], B. subtilis, B. licheniformis and Enterococcus faecium supplemented diets [30], Bacillus spp incorporated diets [32], Biogens® incorporated diets [37] and B. toyoi and B. cereus incorporated diets [34].

Energy utilization performance
The energy utilization performance of LS+SC (4:4) incorporated diet fed group of prawn PL is also paved in Table 2.The feeding rate, absorption rate, conversion rate, NH 3 execratory rate and metabolic rate were found to be higher in PL fed with 2% LS+SC diet, followed by the PL fed with 3%, 4% and 1% diets when compared with control.These differences were found to be statistically significant (P<0.05).Similarly, Seenivasan et al. [6] showed that Binifit TM supplemented diets had improved the energy utilization performance of freshwater prawn, M. rosenbergii PL.Dhanaraj et al. [29] noted that Lactobacillus acidophilus and yeast S. cerevisiae supplemented diets had improved the energy budget of Koi Carp, Cyprinus carpio.It has been reported in pearl spot, Etroplus suratensis fed with Lactobacillus and yeast supplemented diets had significantly improved the feed energy utilization performance [38].It has also been reported in Nile tilapia of the nutrient energy utilization performance was higher in Saccharomyces cerevisiae supplemented diets [39].EL-Haroun et al. [37] showed that Biogen® incorporated diets have improved the growth and feed energy utilization performance of Nile tilapia, Oreochromis niloticus.

Microbial study
The qualitative microbial study showed presence of the rearing control medium and control PL gut following bacteria, such as Bacillus sp., Pseudomonas sp., E. coli, Streptococcus sp., and Klebsiella pneumonia in the Table 3 and 4. In the experimental PL, the presences of Klebsiella pneumonia were replaced by establishment of L. sporogenes (140x10 -4 cfu cells) and S. cerevisiae (90x10 -4 cfu cells) colonies (Table 5).All necessary confirmation biochemical tests were performed and the results are presented (Tables 3 to  6).
Table 3. Biochemical characterization of isolates in control water.Similar results have been reported in the gut of M. rosenbergii PL fed with bioencapsulated L. sporogenes [5].It has been reported in prawn, M. rosenbergii fed with bio-encapsulated L. sporogenes and L. acidophilus that established in the gut [12].It has also been reported that the probiotic bacterial colonies established in the intestine of the

Table 1 .
Ingredients and proximate composition of prepared diets.
Each value is a mean±SD of three replicate analysis, within each row means with different superscripts letters are statistically significant P<0.05 (one way ANOVA and subsequently post hoc multiple comparison with DMRT).S -survival; WG -weight gain; SGR -specific growth rate; FCR -feed conversion rate; FCE -feed conversion efficiency; PERprotein efficiency rate; FR-feeding rate; AR-absorption rate; CR-conversion rate; AE-NH3 excretory rate; MR-metabolic rate