Dietary Prebiotics and Probiotics Influenced the Growth Performance, Feed Utilization and Body

Indices in Snakehead (Channa Striata) Fingerlings

1Mohammad Bodrul Munir*, 1,3Roshada Hashim, 4Mohammad Suhaimee A M and 1,2Siti Azizah Mohd Nor

1School of Biological Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia

2CEMACS, Universiti Science Malaysia, Teluk Bahang, Penang, Malaysia

3Faculty of Science, Universiti Sains Islamic Malaysia, 71800 Bandar Baru Bangi, Malaysia

4FRI Pulau Sayak, 08500 Kota Kuala Muda, Kedah, Malaysia

*Corresponding author: hsjewel730@yahoo.com

Abstract: This study was conducted through a feeding trial with two phases aimed to determine the

influence of selected dietary prebiotics and probiotics on growth performance, feed utilization and

morphological changes; and the duration of their effectiveness for a period of post feeding without any

supplementation. Triplicate groups of fish (22.46g + 0.17) were raised on a feeding trial with six different

treatments respectively three prebiotics- 0.2% β-glucan, 1% glacto-oligosaccharides (GOS), 0.5%

mannan-oligosaccharides (MOS), and two probiotics- 1% live yeast (Saccharomyces cerevisiae),

0.01% Lactobacillus acidophilus (LBA) powder, and a control (non-supplemented diet. All diets contained

40% protein and 12% lipid. Fish were fed to satiation three times daily. No mortalities were recorded in

Phase 1, however 14% was recorded in the control and prebiotic amended fish in Phase 2. At the end of

Phase 1, the growth performance and feed utilization were significantly highest (P<0.05) in the LBA

treated fish, followed by live yeast compared to all the diets tested. Performance of fish on the three

prebiotics diets did not differ significantly more but were significantly better than the control diet. In the

Phase 2 (or post-feeding phase), fish growth continued until the 6th week for the probiotic based diets but

tended to level off for the fish fed the different prebiotic diets after four weeks. Feed Conversion Ratio

(FCR) was higher in all treatments in the post-feeding period. Hepatosomatic Index (HSI) did not differ

significantly among all tested diets. Visceral Somatic Index (VSI) and Intraperitoneal Fat (IPF) were

highest in the LBA based diet and control based diet respectively. The body indices were significantly

changed (P<0.05) between Phase 1 and 2. This study indicates that probiotic based diets have a more

positive influence on the growth, feed utilization and survival of Channa striata fingerlings compared to

supplementation with prebiotics.

Keywords: Prebiotics, Probiotics, Growth Performance, Snakehead (Channa striata)

INTRODUCTION

The striped snakehead, Channa striata (Bloch, 1793), is a carnivorous freshwater fish, which is widely

distributed in Asia. This is a valuable food fish (Wee 1982) contains higher protein (Annasari et al., 2012),

high quality of flesh, low fat, less intramuscular spines and medicinal qualities (Haniffa and Marimuthu,

2004) particularly it’s extracts like fins, scales which are a good source of albumin, and it is traditionally

used to treat injuries, burns. Therefore snakehead aquaculture has recently gained more attention and

the production yield is increased from 16 ton in 1998-2000 to 42 ton in 2010-12 (FAO 2012).

The persistent goal of new world aquaculture (FAO, 2014) is maximizing the efficacy of fish

production with optimizing the profitability. Therefore, the global aquaculture is become more intensified.

This may lead to being high fish yields as well as fish production in per capita area; in contrast it is

directly evolving to deteriorate water quality resultant to out-breaking of fish diseases (Bondad et al.,

2005). Farmers usually control this fish disease using different antibiotics as feed supplements. The

excessive use of antibiotics resultant to develop the antimicrobial resistant pathogens, inhibit or kill the

beneficial microbiota in the gastrointestinal (GI) ecosystem, and finally making antibiotic residue into fish

body that accumulated in fish product to be harmful for human consumption (FAO, 2005). The European

Union banned once to import of fish fed antibiotic feeding supplements on 2006. After that, the

aquaculture scientists, researchers started to explore new strategies substitute of antibiotics in feeding

and health management in fish aquaculture (Balcâzar et al., 2006). They evaluated the new dietary

supplementation (Diana 1997; Abdelghany and Ahmed, 2002) on dietary prebiotics, probiotics,

symbiotics, phytobiotics and other functional dietary supplements (Denev, 2008).

The present study was carried out with similar objective to determine the influence of selective

single dose of dietary prebiotics and probiotics on growth performance, feed utilization & body indices

of Channa striata fingerlings and the duration of their effectiveness for a period of post-feeding without

any supplementation. In general, dietary prebiotics is a non-digestive feed ingredient (Gibson and

Roberfroid 1995) that benefits fish by selectively stimulating growth (Grisdale et al., 2008, Talpur et al.,

2014), while probiotics are live bacteria or cyanobacteria, microalgae, fungi etc. (Fuller, 1989) having

beneficially affects the host growth by improving its intestinal (microbial) balance (Al-Dohail et al., 2009,

Dhanaraj et al., 2010).

METHODOLOGY

Experimental Fish and Husbandry Conditions

The study was conducted at the Aquaculture Research Complex of Universiti Sains Malaysia (USM),

Main Campus, Penang, Malaysia. It was an indoor preliminary study to determine the long term effect of

dietary prebiotics and probiotics feed supplements on snakehead fingerlings growth and health status.

This paper evaluated the effect of dietary prebiotics and probiotics on snakehead fingerlings growth

status only. The study was conducted in two phases without any interval. The first phase was comprised

of 16 weeks while the second phase was the following 8 weeks totalling 24 weeks continuously from the

starting.

A total of 360 snakehead fry (3-4 inch sized) were purchased from the local fish farm, reared for

4 weeks into two outer cemented tanks (2m x 1m x 0.5m) with commercial sea bass pellet feeds

contained 43% crude protein and 6% crude lipid. This was done because of making adaptation with the

environment to save the fish from mortality. Water temperature and pH were recorded twice in a day. The

fishes’ survival was recorded about 80.5%. After 4 weeks, a total of 180 pieces (10 fish/ tank and 3 tanks

for each feeding trial) of snakehead (Channa striata) fingerlings (av. wt. 22.46 g + 0.17) were raised on

experimented diets with control in 18 rounded plastic tanks (200L vol.).

Experimental Diets

In this study, 5 experimental diets along with control (total 6 diets) were prepared at FRI Pulau Sayak,

Kedah and carried to the USM Aquaculture Complex with the air tightened pole-ethylene bags. The diets

were kept at -20ºC frozen temperature. The 5 supplemented diets were 3 prebiotics, 0.2% β-glucan

(Macrogard(R)), 1% glacto-oligosaccharides (Vivinal(R) GOS syrup, Friesland Campina Domo,

Netherland), 0.5% mannan-oligosaccharides (Alltech(R), Actigen 1, USA), and 2 probiotics, 1% live

yeast (Saccharomyces cerevisiae, Alltech(R), YEA-SACC 1026, USA), 0.01%

Lactobacillus acidophilus powder (SigmaR LBA-108 CFU), respectively.

The control diet did not contain any feed supplementation. All the prepared diets were contained

40% protein and 12% lipid. The feed ingredients and proximate composition of diets (Table 1) were

analysed as described in AOAC (1997).

Feeding Trial

There was only one feeding trial conducted with two phases. The first phase was comprised of 16 weeks

with dietary prebiotics and dietary probiotics, followed by another 8 weeks of the control diet in the

second phase. Triplicate groups of fish were raised with the control in indoors 18 tanks (200 litre water

capacity) and fed to satiation three times daily. Water temperature and pH were measured twice daily

(early morning and late afternoon), although these two water parameters were not too much changed

because of indoor closed non flowing continuous aerated water environment, but helped to know about

the cleanness of the tank aquaculture.

Growth performance

Fish weight was taken fortnightly in Phase 1 from the 4th week of the feeding treatment and weekly in

Phase 2. Every feeding treatment had 3 biological replicates and each replicate contained 10 numbers of

Channa striata fingerlings. Before taking the weight of each fish, the water in each replicate tank was

released gradually and then the fish were taken using a soft scoop net and kept the fishes into another

covered container for a while. The fish was taken individually with a small soft towel, dried using tissue

and measured the weight and length; after that the fishes were released to their respective tanks filled by

the clean new water. For analyzing the growth performance, the conditional factor (CF), the relative

growth (RG); the specific growth rate (SGR), survival rate (SR) were determined using different formula

described by Austreng (1978) Busacker et al., (1990) and Ahmed et al., (2002). Moreover, the protein

efficiency rate (PER), food conversion ratio (FCR) was calculated in order to measure the efficiency of

the test feeds in terms of growth for fish using the following formula (Abdel T. et al., 2008; USAID, 2011)

CF(%):{(Final Weight (g) / L3(cm)) x 100}

RG (%): ({Final weight-Initial Weight / initial weight} x 100)

SGR (%): (ln final weight- ln initial weight / nos. of days) x 100

SR (%): {(Final Number of Fish / Initial Number of Fish) x 100}

PER: {(Final Weight-Initial Weight) / Protein Intake}

FCR: (Total Feed Consumption / Weight Gain of Fish)

The hepatosomatic index (HSI), visceral somatic index (VSI) and intraperitonial fat (IPF) were

determined by sacrificing three fishes per replicate tank in each feeding treatment at the end of Phase 1

and Phase 2, respectively, using the following formula (Busacker et al., 1990).

HSI (%): {(Liver Weight / Fish Weight) x 100}

VSI (%): {(Viscera Weight / Fish Weight) x 100}

IPF (%): {(IPF Weight / Fish Weight) x 100}

The fish muscles of 6 feeding treatments were collected in small universal bottles covering with

aluminum foil sheet for measuring the proximate composition. All the aluminum foil covers were punched

and kept it at dry freezer (-70ºC to -75ºC) for 24 hours (non-stop). The freeze dried muscle were taken

out and further analyzed for determining the proximate composition according to the AOAC (1997)

guideline.

Data Analysis

The results were analyzed statistically using SPSS (Version 18). One way ANOVA (Analysis of Variance)

was used to analyse the comparison of the data of growth performance, feed utilization and body indices

in two phases. Multiple comparisons were used with Duncan’s test to analyse the differences between

treatment means at 95% confidence level.

RESULTS

There was a significantly (P<0.05) change observed in the growth of Channa striata fingerlings by

inclusion of dietary prebiotics and probiotics (Table 2) between two phases. The growth performance of

different feeding treatments was significantly increased during the first phase (Table 2), but decreased at

different time significantly (P<0.05) in the second phase. It was always significantly higher in fish fed the

Lactobacillus acidophilus (LBA) diet in both phases. The specific growth rate (SGR) in the 3 prebiotic

treatments did not differ significantly with live yeast (probiotic) during the first phase but lowered

significantly at the end of second phase (Fig 1). Prebiotics and probiotics feed supplements increased the

specific growth rate (SGR) of Channa striata fingerlings (Fig. 1) significantly, it was observed in the first

phase, but it was declined gradually in all prebiotics fishes after 4 weeks in the second phase while no

feed supplement used, followed by live yeast and LBA which dropped after the 6th and 7th week,

respectively (Fig. 1). In both cases LBA was significantly highest (Fig. 1).

The study found that feeding probiotics, particularly LBA, resulted in significantly better feed

utilization efficiency. The FCR and PER were significantly (P<0.05) affected by inclusion of dietary

prebiotics and probiotics (Table 2). In the first phase of the experiment, the lowest feed conversion ratio

(FCR) obtained in the LBA feeding treatments followed by β-glucan; but FCR values for all treatments

increased by the end of the post feeding phase (Table 2). Similarly, protein efficiency rate (PER) was

found higher LBA feeding treatments after 16 weeks followed by β-glucan and GOS treatments; however

during the post feeding trial, the PER was significantly higher in both the probiotics treatments compared

to 3 prebiotics tested (Table 2).

It was an outstanding result that 100% survival was maintained in all feeding treatments till the

end of first phase; however at the end of second phase, it was declined as 90% was in the control and β-

glucan treatments, followed by 88% in MOS and 82% in GOS. Overall 14% mortality was found at the

end of second phase. No mortality was recorded in the probiotics based feeding treatments at both

phases (Table 2).

Condition factor was also affected by the dietary supplement (Fig. 2). Highest significant change

was found in MOS at end of first phase, followed by live yeast, β-glucan, GOS, LBA and control, whereas

no significant difference was found in all prebiotics with the control in post-supplemented feed period or at

the end of second phase. In this period highly significant difference was observed in both probiotics feed

supplements (Fig.2). The study did not find any significance (P<0.05) differences of HSI, VSI and IPF

with the body weight between first and second phase, but it was reducing in post-supplemented feed

period (Fig.2) i.e. second phase.

The proximate compositions of fish muscles (Table 3) were indicated that the compositions were

significantly changed by inclusion of dietary prebiotics and probiotics. The tested diets evolved to

increase significantly the crude protein content, the highest was found in LBA based diet, followed by 3

prebiotics and live yeast (probiotics) compared to the control during end of 16 weeks. In contrast, it was

observed in decrease for crude lipid content and LBA based diet produced low crude lipid in the fish

muscle followed by live yeast, and 3 prebiotics (Table 3). Both phases, the fish muscle contained low

ash, but significantly changed with control diet between two phases.

DISCUSSION

The result obtained from present study revealed that supplementation with dietary prebiotics and

probiotics had a strong effect on growth performance in Channa striata fingerlings. In the first phase, the

performance trend of supplemented diets was LBA>live yeast>β glucan>MOS>GOS (Table 3), non-

supplemented or control treated diet showed the lowest performance. The performance trend

demonstrated clearly that there had some special attributes in the supplemented diets that tended to

enhance the growth performance of Channa striata. Watson and Preedy (2010) stated that dietary

prebiotics and probiotics are the functional bioactive foods which help to promote the growth and health

performance of the living organisms. Both supplements (prebiotics and probiotics) usually modulate the

endogenous flora (probiotic directly) in the gastrointestinal tract producing enzymes or influencing

enzyme activity. The primary role of the digestive tract is to break down foodstuff into small molecules

compatible for absorption across the epithelial border cells of the gastrointestinal tract (Merrifield et al.,

2011) with the aid of the digestive enzymes. The secretion of digestive enzymes can be enhanced in the

intestines of fish by the intake of dietary prebiotics and probiotics. Numerous studies demonstrated that

dietary prebiotics and probiotics are preliminary responsible to modulate the favourable intestinal

microflora that play a major role during secretion of digestive enzymes specially amylase (Xu et al., 2003;

Yanbo and Zirong, 2006; Essa et al., 2010; Askarian et al., 2011; Sang et al., 2011; Wu et al., 2014).

The present study showed the better performance in LBA treated fish compared to other

probiotic, live yeast probable due to have their different mode action in the gastrointestinal tract. Feeding

Lactobacillus acidophilus supplemented diet increases the population of Lactobacillus sp thus not only

replaces the pathogenic bacteria, produce nutrients and stimulate to release the digestive enzymes more

resulting to enhance the digestion process more fast (Cüneyt et al., 2008). On the other hand, the

ingested of live yeast cannot do same action; it involves the maturation of the gut by formation of

colonies. The ability of colonize is thought to be related to cell surface hydrophobicity which helps to grow

the live yeast strains on the intestinal mucous (Wache, 2006). In fact, the different mode of action tended

to make difference of dietary probiotics and probiotics also on growth performance in Channa striata

fingerlings of the present study. The mode of actions in the gastrointestinal tract of the dietary prebiotics

tested in this study was indirect. It is likely that the probiotics, which are live bacteria or fungi (Fuller,

1989), have a pro-bioactive role (i.e. bio-active originating from food matrix and bacteria) in the

gastrointestinal wall resulting in the enhancement of the fermentation rate in the intestinal colon (Gill,

1998). Growth performance responded to the ingestion of dietary prebiotics showed different probable

due to their structural differences. β-glucan tested in this study was an unbranched homopolysaccharides

structure, where as other two feed supplements, MOS and GOS were the branched

heteropolysaccharides structure. The unbranched homopolysaccharides contain the same

monosaccharides like glucose; however the branched heteropolysaccharides contain different

monosaccharides linked with the glycosidic bonds in nature (Chanpul et al., 2012). Although the

structural differences tended to make difference of efficacy of the three prebiotics, however the result

showed in the present study was not more significant difference. The probable cause is β-glucans are

proven as an active biological response modifier prebiotic, is a soluble carbohydrates (Bhon, 1995)

obtained from the cell walls of live yeast (Saccharomyces cerevisiae), while Glacto-oligosaccharides

(GOS) and mannan-oligosaccharides (MOS) contain oligosaccharides carbohydrates with low molecular

weight with a degree of polymerization value (Sander et al., 2005 and Roberfoid & Slavin, 2000). Overall,

the results obtained from the first phase of this study revealed a positive effect of dietary prebiotics and

probiotics as feed supplements to Channa striata fingerlings. The survival data of the present study also

demonstrated the same information relevant to the growth performance. This result was supported by

the previous study of Talpur et al.,(2014) who conducted with a selective single dose of dietary prebiotics

and probiotics as feed supplements for the study of Channa striata fingerlings. The same evidence was

observed on African cat fish, Clarias garepinus (Al-Dohail et al., 2009) and Cyprinus carpio (Dhanaraj et

al., 2010), hybrid striped bass (Li and Gatlin, 2005), rainbow trout (Staykove et al., 2007), European sea

bass (Torrecillas et al., 2007) and red drum, sciaenops acellatus (Zhou et al., 2010) respectively. Similar

to the result of growth performance in Phase 1, the feed utilization and the body indices of Channa striata

were also affected positively by the inclusion of dietary prebiotics and probiotics (Table 3). All the tested

diets reduced the FCR at below 2 including the control diet probable due to contain 40% protein and 12%

lipids. The bioactive attributes of dietary prebiotics and probiotics accelerated to reduce more FCR, which

indicated that the tested diets were viable economically. Besides, the inclusion of dietary prebiotics and

probiotics increased the protein efficiency rate, which was good indicator, because it helped to reduce the

FCR (USAID, 2011). Fish fed with LBA was found significantly best, followed by live yeast, which was

another probiotics as beneficial fungi. The tested LBA and fungi may lead to active more in

gastrointestinal tract (Marteau et al., 1993) resultant to increase the protein efficiency rate and to reduce

the FCR. In contrast, the tested three dietary prebiotics feeding supplements were created space for

facilitating the beneficial bacteria, and by nature they are very similar to low digestible carbohydrates and

they influence the osmotic pressure in the gastrointestinal tract until fermentation (Roberfoid and Slavin,

2000) resulting to enhance to the endogenous bacteria like bacillus and the intestinal gas production for

more digestive activities. Therefore it led to decrease the FCR with increasing the PER.

The present study also revealed that the inclusion of dietary prebiotics and probiotics led to keep

well the conditional factor of its growth, because it indicates the nutritional state of an individual fish

(Schreck and Moyle, 1990). The proximate composition analysis indicated that fish muscle of this study

contained high protein, but low fat and ash. Usually, Channa striata is a fresh water fish which contains

high protein (Annasari et al., 2012) and low fat. The inclusion of dietary prebiotics and probiotics

compared with the control of this study led to enhance more crude protein and less lipid contents that

may good for food fish (Wee 1982).

The addition of post feeding trial (Phase 2) where the treated fishes were fed with non-

supplemented diets or control for a time being registered the present study a complete study about the

effect of dietary prebiotics and probiotics on fish growth performance. The present study has demanded

that this post feeding trial was so far first time studies in any kind of fish nutrition research. The

performance trend of specific growth rate (SGR) tended to show clear difference between phase 1 and

phase 2 in the present study. In the post feeding phase, it appears that the bioactive role remains

constant 7 weeks for LBA, 6 weeks for live yeast (Fig. 1) and 4 weeks for all three prebiotics tested in this

study. The probable cause of this incidence was the effect of residue which stored in the gastrointestinal

tract. In Phase 1, when the fish were fed with the supplemented diets, fishes may not use all the nutrients

derived from these diets for growth purposes; besides 16 weeks continuous supplemented feeding

treatments in Phase 1 enriched the deposition of supplemented diets as residue which might work in the

Phase 2 where the treated fishes were fed only control diet. This statement has become true when the

SGR of supplemented diets was compared to the control amended fish since Phase 1 (Fig 1). The

residual effects supplanted amended fish in Phase 2 or post-feeding trial was reflected to the higher of

FCR and the lower PER. The argument is fish needs the same energy to maintain it’s growth similar to

Phase 1; but replacing the supplemented diets with the control (non-supplemented diets) could not bear

the same energy for maintain the growth performance. Therefore, the growth performance of

supplemented amended fishes was dropped periodically in Phase 2. This statement is eventually proved

if compare the survival percentage of fish between phase 1 and phase 2. Nevertheless, there were no

more significant morphological changes (HSI, VSI and IPF) in fish between these two phases probable

due to have no effect on biological changes whether it stopped.

In conclusion, the result obtained of the present study established the positive efficacy of

supplemented diets. The studied fish growth, low FCR, high PER with low fat led to prove that the

formulated fish feed with dietary prebiotics and probiotics supplementation had a positive effect,

particularly supplementation with 0.01% (108 CFU) Lactobacillus acidophilus (LBA) powder, which led to

fish growth highest with low FCR and high PER. However this was a preliminary study; need to be

studied in depth with other parameters like nutrient digestibility, blood parameter, gut microflora and

innate immune response status etc. for Channa striata fingerlings.

ACKNOWLEDGEMENT

The authors would like to express the thanks to FRGS (Ref: 203/PBIOLOGI/6711308) as well as USM

Global Fellowship for the financial support to conduct the research. Special thanks goes

to FRI Pulau Sayak, Kedah for proving the facility of experimental diets preparation, AllTech(R) for

providing free of cost (for research) of the Bioactin and Yaa-Sac, as well as to similar to

FriedlandCampina Domo(R) for Vivinal GOS Syrup & Bio-Origin for Macroguard(R) β-glucan.

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CT=Control Diet without supplementation; BG= Diet with β-Glucan supplement; GS= Diet with GOS

supplement; MS= Diet with MOS supplement; YS=Diet with Live Yeast supplement; LB=Diet with

Lactobacillus acidophillus supplement

Table 1: Feed Ingredients and Proximate Composition of the Formulated Diet (g/ kg, dry matter). Ingredients Control β-Glucan 0.2% GOS 1% MOS 0.5% Live Yeast 1% L.acidophils 0.01%

Danish Fish Meala 534 534 534 534 534 534

Korean Corn Starch 340 340 340 340 340 340

Fish oil 5 5 5 5 5 5

Soyabean oil 60 60 60 60 60 60

Cellulose 11 8 1 6 1 10.9

CMCb 10 10 10 10 10 10

Vitamins mixc 20 20 20 20 20 20

Minerals mixd 20 20 20 20 20 20

Supplement 0 2 10 5 10 0.1

Proximate composition g/kg Control β-Glucan 0.2% GOS 1% MOS 0.5% Live Yeast 1% L.acidophils 0.01%

Moisture 81.9 52.2 63.1 71.9 96.5 92.8

Protein 410.0 407.3 409.4 406.8 409.1 409.7

Lipid 118.8 117.5 118.4 118.0 120.3 121.2

Ash 10.1 10.2 9.8 10.3 9.9 10.6

Fiber 123.0 123.2 123.2 121.8 121.8 120.6

NFCe 256.2 289.6 276.1 271.2 242.4 245.1

GEf (MJ/kg) 198.9 197.6 198.5 199.2 198.7 196.9

aDanish Fish Meal Kg-1 =Crude Protein 746.6 and Crude Lipid 101.6

bCMC= Carboxymethyl Cellulose

cVitamin Mix Kg-1 = Rovimix 6288, Roche Vitamins Ltd. Switzeland; Vit A 50 million i.u., Vit D 310 million i.u., Vit E 130 g, Vit B1 10g,

Vit B2 25g, Vit B6 16g, Vit B12 100 mg, Biotin 500mg, Pantothenic acid 56g, Folic Acid 8g, Niacin 200g, Anticake 20g, Antioxident

200mg, Vit K3 10g and Vit C 35g

dVitamin Mix Kg-1 = Calcium phosphate (monobasic) 397.65g, Calcium lactate 327g, Ferous sulphate 25g, Magnessium sulphate

137g, Potassium chloride 50g, Sodium chloride 60gm, Potassium iodide 150mg, Copper sulphate 780mg, Manganese oxide

800mg, Cobalt carbonate 100mg, Zinc oxide 1.5g and Sodium selenite 20g.

eNFE = Nitrogen Free Extract (1000-{Moisture+Protein+Lipid+Ash+Fiber})

fGE =Gross Energy Measured using Bomb Calorimeter, Parr 1356 Bomb Calori.

Table 2: Growth performance, feed utilization and survival of Channa striata fingerlings.

Parameters Control β-Glucan GOS MOS Live Yeast LBA

Initial Weight (g)

Initial 22.34+0.05 22.45+0.17 22.57+0.13 22.30+0.21 22.57+0.13 22.47+0.16

Weight Gain (g)

Phase 1 32.21+0.55 a 58.15+0.32 b 58.18+0.27 b 58.64+0.36 bc 59.56+0.57 c 71.39+0.89 d

Phase 2 48.00+0.10 a 75.77+0.61 c 76.20+0.30 c 73.43+0.65 b 89.40+0.70 d 112.90+0.65 e

RG (%)

Phase 1 44.16+2.16a 159.00+0.97bc 157.76+2.18b 163.00+2.10cd 163.91+4.07c 217.68+2.83d

Phase 2 114.86+0.37a 237.44+0.15c 237.57+1.00c 229.30+0.90b 296.10+2.71d 402.38+1.86e

SGR (%)

Phase 1 0.33+0.01 a 0.85+0.03bc 0.84+0.01 b 0.86+0.07c 0.87+0.01 c 1.03+0.01 d

Phase 2 0.46+0.00a 0.72+0.00c 0.72+0.00c 0.71+0.00b 0.82+0.00 d 0.96+0.00 e

FCR

Phase 1 1.90+0.17d 1.63+0.06b 1.80+0.00cd 1.73+0.06bc 1.64+0.006b 1.43+0.06a

Phase 2 1.79+0.00d 1.76+0.00c 1.89+0.00f 1.82+0.00e 1.80+0.00cd 1.56+0.01a

PER

Phase 1 1.28+0.10a 1.50+0.08c 1.33+0.01ab 1.42+0.01bc 1.50+0.04c 1.71+0.04d

Phase 2 1.40+0.00b 1.40+0.00b 1.30+0.00a 1.30+0.02a 1.38+0.16ab 1.56+0.06d

Survival

Phase 1 100% 100% 100% 100% 100% 100%

Phase 2 90% 90% 82% 88% 100% 100%

Each column represents different feeding treatments. All values are mean +SD obtained from three replicates groups (n=3); Data

with different superscripts in the same row indicate significant differences (P<0.05) among the feeding treatments. RG, Relative

Growth; SGR, Specific Growth Rate; FCR, Feed Conversion Rate; PER, Protein Efficiency Rate; β Glucan, Beta glucan; GOS,

Glacto-oligosaccharides; MOS, Manna-oligosaccharides; Live Yeast, Saccharomyces cerevisiae; LBA, Lactobacillus acidophilus.

Table 3: Proximate composition of body muscle between Phase 1 and Phase 2.

Parameters Control β-Glucan GOS MOS Live Yeast LBA

Moisture (%) Phase 1 5.24+0.12d 4.37+0.40c 4.52+0.29c 4.57+0.22c 3.57+0.38b 1.69+0.29a

Phase 2 2.52+0.45a 3.70+0.40c 2.38+0.02b 2.73+0.28ab 2.57+0.42a 3.29+0.44bc

Crude Protein (%) Phase 1 81.13+0.54a 86.80+0.71b 86.56+0.37b 85.92+0.36b 86.19+0.41b 90.53+0.57c

Phase 2 85.39+0.25ab 84.45+0.38a 86.12+0.11b 86.15+0.66b

85.13+0.40ab 85.92+0.97b

Crude Lipid (%) Phase 1 6.92+0.07d 5.49+0.10bc 5.52+0.01c 5.61+0.09c 5.36+0.04ab 5.25+0.12a

Phase 2 6.61+0.22c 6.43+0.50bc 6.05+0.07bc 5.27+0.12a 6.28+0.45bc 5.88+0.04b

Ash (%) Phase 1 5.34+0.08f 2.19+0.08b 2.59+0.27c 3.04+0.09d 4.09+0.04e 1.59+0.32a

Phase 2 5.07+0.41 5.01+0.06 5.18+0.20 5.40+0.55 5.63+0.49b 4.64+0.53a

Each column represents different feeding treatments. All values are mean +SD obtained from three replicates groups (n=3); Data

with different superscripts in the same row indicate significant differences (P<0.05) among the feeding treatments; β Glucan, Beta

glucan; GOS, Glacto-oligosaccharides; MOS, Manna-oligosaccharides; Live Yeast, Saccharomyces cerevisiae; LBA, Lactobacillus

acidophilus.